Pathogen-inactivated red blood cell compositions

ABSTRACT

The present invention provides pathogen-inactivated red blood cell compositions suitable for infusion into a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/936,763, now U.S. Pat. No. 8,900,805, submitted under 35 U.S.C. §371as a U.S. National Stage Application of International Application No.PCT/US2009/040032 filed Apr. 9, 2009, which claims priority benefit ofU.S. Provisional Application No. 61/043,666, entitled “Quenching MethodsFor Red Blood Cell Pathogen Inactivation” filed Apr. 9, 2008, and U.S.Provisional Application No. 61/087,034, entitled “Quenching Methods ForRed Blood Cell Pathogen Inactivation” filed Aug. 7, 2008. Thedisclosures of each of these applications are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The field of this invention relates to improved methods of quenchingreactive electrophilic compounds used in treating blood products toinactivate possible pathogen contaminants. In particular, nucleophiliccompounds, such as thiols, are used at an elevated concentration toquench the reactive electrophilic compounds in red blood cellcompositions, then decreased in concentration to reduce red blood cell(RBC) dehydration.

BACKGROUND OF THE INVENTION

The transmission of disease by blood products and other biologicalmaterials remains a serious health problem. While significant advancesin blood donor screening and blood testing have occurred, viruses suchas hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiencyvirus (HIV) may escape detection in blood products during testing due tolow levels of virus or viral antibodies. In addition to the viralhazard, there are currently no adequate licensed tests to screen for thepresence of non-viral microbes, such as bacteria or protozoans, in bloodintended for use in transfusions. The risk also exists that a hithertounknown pathogen may become prevalent in the blood supply and present athreat of disease transmission, as in fact occurred before therecognition of the risk of HIV transmission via blood transfusions.

Chemical agents have been introduced into blood or blood plasma toinactivate pathogens prior to clinical use of the blood product.Typically, for blood products having little or no red blood cellcontent, such as platelets and plasma, photochemically activatedcompounds such as psoralens are used. For red blood cell-containingblood products, compounds have been developed for pathogen inactivation,which do not require photoactivation. These compounds typically haveelectrophilic groups that react with pathogens, more specifically withpathogen nucleic acid. For example, U.S. Pat. No. 5,055,485 describesthe inactivation of viruses in cell and protein-containing compositionsusing aryl diol epoxides. Other compounds that generate electrophiles insitu may be used. LoGrippo et al. evaluated the use of nitrogen mustard,CH₃—N(CH₂CH₂Cl)₂, for viral inactivation. LoGrippo et al., Proceedingsof the Sixth Congress of the International Society of Blood Transfusion,Bibliotheca Haematologica (Hollander, ed.), 1958, pp. 225-230. Moresignificantly, U.S. Pat. Nos. 5,691,132; 6,177,441; 6,410,219;6,143,490; and 6,093,725, the disclosures of which are herebyincorporated by reference, describe the use of compounds that have anucleic acid targeting component as well as an electrophilic componentthat reacts with the nucleic acid in order to inactivate the pathogen.U.S. Pat. Nos. 6,093,725 and 6,514,987, the disclosures of which arehereby incorporated by reference, describe similar compounds, whereinthe nucleic acid targeting component of the compound is linked to thereactive electrophilic component via a hydrolysable linker. U.S. Pat.Nos. 6,136,586 and 6,617,157, the disclosures of which are herebyincorporated by reference, describe using ethyleneimine oligomers andrelated compounds for pathogen inactivation. These ethyleneimine-derivedcompounds typically have an aziridine group, which provides the reactiveelectrophilic component, and a polyamine component, which providesnucleic acid targeting of the compound. The general class of nucleicacid targeted compounds having an electrophilic or similar groupreactive with the nucleic acid are used to inactivate pathogens inblood, blood products, and a variety of samples of biological origin.

There is some concern that, while these compounds are designed tospecifically target nucleic acids, they may react with other componentsof the blood, such as proteins or cellular membranes. Theseside-reactions are unfavorable and may cause adverse effects, such asmodifications of proteins and cell membranes that may be recognized bythe immune system. When such treated blood products are used repeatedly,they may result in an immune response of the recipient to the treatedblood product. U.S. Pat. Nos. 6,270,952; 6,709,810; and 7,293,985, thedisclosures of which are hereby incorporated by reference, describemethods of quenching such pathogen-inactivating compounds in order toreduce the level of any such adverse side-reactions. U.S. PatentPublication No. 2006/0115466, the disclosure of which is herebyincorporated by reference, describes improvements to these quenchingconditions which address an immune response developed against thepathogen-inactivating compound. However, despite the improvement inquenching effectiveness, in some cases the treated red blood cells havebeen found to have decreased lifespan attributed to increased celldehydration as measured by decreased osmotic fragility and decreasedspun hematocrit.

Thus, there is a need for methods to reduce unwanted electrophilicside-reactions of pathogen-inactivating compounds while preserving theability of the pathogen-inactivating compound to inactivate harmfulpathogens without adversely affecting the vitality and lifespan of thetreated blood product. Specifically, there is a need for improvedmethods of quenching pathogen-inactivating compounds in red blood cells.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a variety of methods for treating redblood cell compositions with a pathogen-inactivating compound and aquencher under conditions which provide suitable pathogen inactivationand reduction of unwanted side reactions (such as modification of thered blood cells), while reducing or minimizing adverse effects such asincreased dehydration of the cells. In some embodiments, the quencher isglutathione which is neutralized with an appropriate amount of base. Insome embodiments, the method involves reducing the concentration ofglutathione following pathogen inactivation.

In one aspect, the present invention provides a method of treating a redblood cell composition comprising: a) providing i) an effective amountof a pathogen-inactivating compound to inactivate a pathogen, ifpresent, wherein the pathogen-inactivating compound comprises afunctional group which is, or which forms, a reactive electrophilicgroup, ii) an effective amount of a quencher comprising a thiol group,wherein the thiol is capable of reacting with the reactive electrophilicgroup of the pathogen-inactivating compound, and iii) a compositioncomprising red blood cells; b) mixing the pathogen-inactivating compoundand quencher with the composition comprising red blood cells; and c)sufficiently decreasing the concentration of the quencher in the mixtureto an amount which reduces the level of red blood cell dehydrationresulting from storage of the mixture, relative to the level of redblood cell dehydration resulting from storage of the mixture at theoriginal concentration of quencher. In some of these embodiments,decreasing the concentration of the quencher comprises removal of thesolution used during inactivation and addition of a final additivesolution (e.g., any solution described in herein, such as SAG-M, AS-5 orany solution of Tables 2, 3, or 4).

In some embodiments, step (a) further comprises providing a suitablebase, step (b) further comprises mixing the base with the compositioncomprising red blood cells, and the base is of sufficient amount toreduce the level of an unwanted reaction of the pathogen-inactivatingcompound with red blood cells in the mixture, relative to the mixturewithout the base. In some embodiments, the unwanted reaction of thepathogen-inactivating compound with red blood cells is modification ofthe surface of the red blood cells by the pathogen-inactivatingcompound. In some embodiments, step (a) further comprises providing asuitable base, step (b) further comprises mixing the base with thecomposition comprising red blood cells, and the base is of sufficientamount to reduce the level of anti-pathogen inactivating compoundantibody binding to the treated red blood cell composition in theresulting mixture by at least about 5% (or at least about 10%, at leastabout 25%, at least about 50%, at least about 75%, or at least about90%) relative to the mixture without the base. In some embodiments, thebase and the quencher are mixed with the red blood cell compositionprior to, at the same time, or no more than about 30 minutes aftermixing the pathogen-inactivating compound with the red blood cellcomposition. In some embodiments, the base and the quencher are mixedtogether prior to mixing either the base or the quencher with the redblood cell composition. In some embodiments, the base is NaOH. In someembodiments, the base is a basic buffer. In some embodiments, the basecomprises about 0.5 to 1.5 equivalents of base, where an equivalentmeans a molar amount that is equivalent to the molar amount of quencherin the mixture. In some embodiments, the base comprises about 0.75 to1.25 equivalents of base. In some embodiments, the base comprises about1 equivalent of base. In some embodiments, the resulting mixture of step(b) has a pH at 37° C. of about 6.0 to 7.5. In some embodiments, the pHis about 6.5 to 7.1. In some embodiments, the pH is about 6.8 or 6.9.

In some embodiments, the quencher comprises cysteine or a derivative ofcysteine. In some embodiments, the quencher is glutathione or apharmaceutically acceptable salt thereof. In some embodiments, theconcentration of the quencher in the resulting mixture of step (b) isgreater than 2 mM. In some embodiments, the quencher concentration isabout 5 mM to about 30 mM. In some embodiments, the quencherconcentration is about 15 mM to about 25 mM. In some embodiments, thequencher concentration is about 20 mM.

In some embodiments, decreasing the concentration of quencher in step(c) comprises centrifugation of the mixture followed by removal of thesupernatant. In some embodiments, step (c) comprises size-exclusionseparation. In some embodiments, the quencher in the resulting mixtureof step (c) is at a concentration of less than about 10 mM. In someembodiments, the quencher concentration is less than about 8 mM. In someembodiments, the quencher concentration is less than about 6 mM (or lessthan about 4 mM, or less than about 2 mM). In some embodiments, storageof the mixture is storage of the mixture for greater than 10 days at 4°C. In some embodiments, storage of the mixture is storage of the mixturefor greater than 42 days (or 28 days) at 4° C. In some embodiments, themethod comprises the addition of an additive solution (e.g., anyadditive solution described in Table 2, and/or an additive solutioncomprising sodium chloride, adenine, glucose, phosphate, guanosine,citrate and/or mannitol). In some embodiments, the mixture is stored inan additive solution (e.g., any additive solution described in Table 2,and/or an additive solution comprising sodium chloride, adenine,glucose, phosphate, guanosine, citrate and/or mannitol). In someembodiments, the method further comprises replacement of a treatmentsolution (e.g., any solution described in Tables 2, 3, or 4 and/or asolution comprising sodium chloride, adenine, glucose, phosphate,guanosine, citrate, and/or mannitol) with an additive solution (e.g.,any additive solution described in Table 2, and/or an additive solutioncomprising sodium chloride, adenine, glucose, phosphate, guanosine,citrate and/or mannitol). In some embodiments, the chlorideconcentration of the composition prior to decreasing the concentrationof the quencher is less than about 100 mM (or about 75 mM). In someembodiments, the chloride concentration of the composition followingdecreasing the concentration of the quencher and/or adding the additivesolution is greater than about 100 mM (or about 125 mM).

In some embodiments of each of the aforementioned methods, as well asother methods described herein, the functional group is a mustard, amustard intermediate, or a mustard equivalent. In some embodiments, thefunctional group is, or is capable of forming, an aziridinium ion. Insome embodiments, the reactive electrophilic group is capable ofreacting with nucleic acids. In some embodiments, thepathogen-inactivating compound further comprises a nucleic acid bindingligand. In some embodiments, the nucleic acid binding ligand is anintercalator. In some embodiments, the intercalator is an acridine. Insome embodiments, the pathogen-inactivating compound comprises afrangible linker linking the functional group and the nucleicacid-binding ligand. In some embodiments, the pathogen-inactivatingcompound is β-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethy)amino]ethylester. In some embodiments, the concentration of the pathogeninactivation compound in the resulting mixture of step (b) is about 0.1μM to about 5 mM. In some embodiments, the concentration is sufficientto inactivate at least 1 log of a pathogen in the red blood cellcomposition, if present. In some embodiments, the concentration issufficient to inactivate at least 3 logs of a pathogen. In someembodiments, the time between step (b) and step (c) is about 1 to 48hours. In some embodiments, the time is about 10 to 30 hours. In someembodiments, the time is about 15 to 25 hours. In some embodiments, thetreatment inactivates at least 1 log of a pathogen contaminant in thered blood cell composition, if present. In some embodiments, thetreatment inactivates at least 3 logs. In some embodiments, the methodfurther comprises the step of decreasing the concentration of thepathogen-inactivating compound in the mixture. In some embodiments, thesteps of decreasing the concentration of the quencher in the mixture anddecreasing the concentration of the pathogen-inactivating compound inthe mixture occur at the same time.

In some embodiments of each of the aforementioned methods, as well asother methods described herein, at 20 hours following step (b), the redblood cells (RBCs) of the resulting mixture have an anti-pathogeninactivating compound antibody binding capacity (ABC) of less than 65%compared to the ABC value of red blood cells from the same method underthe same conditions, but without the use of base. In some embodiments,the RBCs have an average ABC of less than about 50,000. In someembodiments, the RBCs have an average ABC of less than about 40,000. Insome embodiments, the RBCs have an average ABC of between about 25,000and 70,000. In some embodiments, the RBCs have an average ABC of betweenabout 35,000 and 45,000. In some embodiments, the RBCs of the resultingmixture have less then 1% hemolysis following step (c). In someembodiments, the RBCs have less then 1% hemolysis at a time of 42 days(or 28 days) at 4° C. following step (c). In some embodiments, the RBCsof the resulting mixture have a Packed Cell Volume (PCV) of greater than50% following step (c). In some embodiments, the RBCs have a PCV ofgreater than 50% at a time of 42 days (or 28 days) at 4° C. followingstep (c). In some embodiments, the RBCs of the resulting mixture have aMedian Corpuscular Fragility value greater than 140 (or 150) after 42days (or 28 days) at 4° C. following step (c). In some embodiments, theamount of pathogen inactivation compound is not reduced and/or thepathogen inactivation compound is not contacted with a compoundadsorption device (CAD).

In an additional aspect, the invention provides a method of reducingdehydration of red blood cells, comprising: a) providing a red bloodcell composition comprising a mixture of i) a quencher, where thequencher is capable of reacting with a pathogen-inactivating compound,and ii) red blood cells; and b) sufficiently decreasing theconcentration of the quencher (and optionally the concentration of thepathogen-inactivating compound and/or byproducts thereof) in the mixtureto an amount which reduces the level of red blood cell dehydrationresulting from storage of the mixture relative to the level of red bloodcell dehydration resulting from storage of the mixture at the originalconcentration of quencher. In some embodiments, the quencher comprisescysteine or a derivative of cysteine. In some embodiments, the quencheris glutathione or a pharmaceutically acceptable salt thereof. In someembodiments, the quencher in the resulting mixture of step (b) is at aconcentration of less than about 10 mM. In some embodiments, thequencher concentration is less than about 8 mM. In some embodiments, thequencher concentration is less than about 6 mM, or less than about 2 mM.In some embodiments, the red blood cells (RBCs) of the resulting mixturehave less then 1% hemolysis following step (b). In some embodiments, theRBCs have less then 1% hemolysis at a time of 42 days (or 28 days) at 4(C following step (b). In some embodiments, the RBCs of the resultingmixture have a Packed Cell Volume (PCV) of greater than 50% followingstep (b). In some embodiments, the RBCs of the resulting mixture have aPCV of greater than 50% at a time of 42 days (or 28 days) at 4 (Cfollowing step (b). In some embodiments, the RBCs of the resultingmixture have a Median Corpuscular Fragility value greater than 140 (or150) after 42 days (or 28 days) at 4° C. following step (b). In someembodiments, storage of the mixture is storage of the mixture forgreater than 10 days at 4° C. In some embodiments, storage of themixture is storage of the mixture for greater than 42 days (or 28 days)at 4° C. In some embodiments, the amount of pathogen inactivationcompound is not reduced and/or the pathogen inactivation compound is notcontacted with a compound adsorption device (CAD).

Red blood cell (RBC) compositions produced by each of the aforementionedmethods are provided. RBC compositions preparable by each of theaforementioned methods are also provided.

In a further aspect, the invention provides a composition comprising a)red blood cells, wherein the red blood cells have covalently reactedwith an electrophilic group of a pathogen-inactivating compound; and b)a quencher comprising a thiol group that is capable of reacting with thepathogen-inactivating compound; wherein the composition is suitable forinfusion into humans after storage up to 42 days (or 28 days) at 4° C.In some embodiments, at least 1 log of a pathogen is inactivated, ifpresent. In some embodiments, at least 3 logs are inactivated. In someembodiments, the electrophilic group is a mustard, a mustardintermediate, or a mustard equivalent. In some embodiments, theelectrophilic group is, or is capable of forming, an aziridinium ion. Insome embodiments, the electrophilic group is capable of reacting withnucleic acids. In some embodiments, the electrophilic group iscovalently reacted with the cell surface of the red blood cells. In someembodiments, the pathogen-inactivating compound further comprises anucleic acid binding ligand. In some embodiments, the nucleic acidbinding ligand is an intercalator. In some embodiments, the intercalatoris an acridine. In some embodiments, the pathogen-inactivating compoundcomprises a frangible linker linking the electrophilic group and thenucleic acid binding ligand. In some embodiments, thepathogen-inactivating compound is β-alanine, N-(acridin-9-yl),2-[bis(2-chloroethy)amino]ethyl ester. In some embodiments, the quenchercomprises cysteine or a derivative of cysteine. In some embodiments, thequencher is glutathione or a pharmaceutically acceptable salt thereof.In some embodiments, the quencher is at a concentration that issufficiently low to avoid or minimize red blood cell dehydration duringstorage. In some embodiments, the quencher concentration is less thanabout 10 mM. In some embodiments, the quencher concentration is lessthan about 8 mM. In some embodiments, the quencher concentration is lessthan about 6 mM, or less than about 2 mM. In some embodiments, the redblood cells (RBCs) have a Packed Cell Volume (PCV) of greater than 55%.In some embodiments, the RBCs have a PCV of greater than 60%. In someembodiments, the RBCs have an average antibody binding capacity (ABC) ofless than about 50,000. In some embodiments, RBCs have an average ABC ofless than about 40,000. In some embodiments, the RBCs have an averageABC of between about 25,000 and 60,000. In some embodiments, the RBCshave an average ABC of between about 25,000 and 70,000. In someembodiments, the RBCs have an average ABC of between about 35,000 and45,000. In some embodiments, the composition further comprises anadditive solution (e.g., any additive solution described in Table 2,and/or an additive solution comprising sodium chloride, adenine,glucose, phosphate, guanosine, citrate and/or mannitol). In someembodiments, the chloride concentration of the additive solution and/orof the composition is greater than about 100 mM (or about 125 mM).

In an additional aspect, the invention provides methods of infusing redblood cells into an individual, comprising: a) providing any one of theaforementioned red blood cell compositions or a red blood cellcomposition produced by any one of the methods described herein and, b)infusing the red blood cell composition into the individual.

In one aspect, the invention provides a method of treating a red bloodcell composition comprising: (a) mixing (i) a pathogen-inactivatingcompound comprising a functional group which is, or which forms, areactive electrophilic group (e.g., an effective amount of apathogen-inactivating compound to inactivate a pathogen, if present);(ii) a quencher (e.g., an effective amount of a quencher) comprising athiol group, wherein the thiol is capable of reacting with the reactiveelectrophilic group of the pathogen-inactivating compound; and (iii) acomposition comprising red blood cells; and (b) sufficiently decreasingthe concentration of the quencher in the mixture to an amount whichreduces the level of red blood cell dehydration resulting from storageof the mixture relative to the level of red blood cell dehydrationresulting from storage of the mixture at the original concentration ofquencher. In some of these embodiments, decreasing the concentration ofthe quencher comprises removal of the solution used during inactivationand addition of a final additive solution (e.g., any solution describedin herein, such as SAG-M, AS-5 or any solution of Tables 2, 3, or 4).The method may comprise any one or more of the embodiments listed aboveand/or herein.

In some embodiments, the method further comprises mixing a suitable basewith the composition comprising red blood cells, and the base is ofsufficient amount to reduce the level of an unwanted reaction of thepathogen-inactivating compound with red blood cells in the mixture,relative to the mixture without the base. In some embodiments, theunwanted reaction of the pathogen-inactivating compound with red bloodcells is modification of the surface of the red blood cells by thepathogen-inactivating compound. In some embodiments, method furthercomprises mixing a suitable base with the composition comprising redblood cells, and the base is of sufficient amount to reduce the level ofanti-pathogen inactivating compound antibody binding to the treated redblood cell composition in the resulting mixture by at least about 5% (orat least about 10%, at least about 25%, at least about 50%, at leastabout 75%, or at least about 90%) relative to the mixture without thebase.

In one aspect, the invention provides a method of reducing dehydrationin a red blood cell composition wherein the composition is a mixturecomprising a quencher capable of reacting with a pathogen-inactivatingcompound, and red blood cells; the method comprising, sufficientlydecreasing the concentration of the quencher in the mixture to an amountwhich reduces the level of red blood cell dehydration resulting fromstorage of the mixture relative to the level of red blood celldehydration resulting from storage of the mixture at the originalconcentration of quencher.

In another aspect, the invention provides a method of red blood cellsinfusion, comprising infusing a red blood cell composition describedherein into an individual.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the osmotic fragility of red blood cells at various levelsof base after initial quencher dosing as described in Example 6.

FIG. 2 shows the red blood cell density at various levels of base at 20hours of incubation.

FIG. 3 shows the red blood cell density at various levels of base afterincubation and 36 days of storage.

FIG. 4 shows the osmotic fragility of red blood cells after incubationand 36 days of storage with and without decreasing the quencherconcentration (i.e. with/without exchange step).

FIG. 5 shows the osmotic fragility of red blood cells after incubationand 42 days of storage with varying initial quencher concentration (withexchange step) compared to moderate initial quencher concentration(without exchange step).

FIG. 6 shows the osmotic fragility of red blood cells after incubationand 36 days of storage with and without pathogen-inactivating compound.

FIG. 7 shows the antibody binding capacity (ABC) values for several redblood cell preparations using the methods of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for treating red blood cellcompositions to inactivate pathogens which may be present, whilereducing or minimizing unwanted side reactions (such as modification ofthe red blood cells leading to an undesired immune response) and whilereducing or minimizing adverse effects on cell vitality (e.g., decreasedosmotic fragility and/or increased dehydration) and/or lifespan duringand after treatment. We have found that the proper control of pH inconjunction with suitable amounts of quencher during the pathogeninactivation process can reduce initial dehydration of red blood cellstreated with a pathogen-inactivating compound. The process can then befollowed by reduction of the initial quencher concentration to providehealthy pathogen-inactivated red blood cells capable of cell storage,without significant changes in osmotic fragility. These methods areparticularly suitable for preparation of red blood cell compositions inwhich pathogens have been inactivated for clinical use, especially whenthe compositions are to be stored for a period of time prior to clinicaluse.

Accordingly, the present invention in one aspect provides a method oftreating a red blood cell composition comprising a pathogen-inactivatingcompound and a quencher, by (1) mixing the pathogen-inactivatingcompound and quencher with the composition comprising red blood cells;and (2) sufficiently decreasing the concentration of the quencher toreduce the level of red blood cell dehydration resulting from storage ofthe mixture relative to the level of red blood cell dehydrationresulting from storage of the mixture at the original concentration.

In other aspects, the present invention provides methods of reducingdehydration and/or increasing osmotic fragility in red blood cells, aswell as methods of infusing red blood cells into humans. Also providedare treated red blood cell compositions.

Red Blood Cells

Red blood cell compositions of the invention include, but are notlimited to, any blood product comprising red blood cells (e.g., humanblood), wherein the blood product provides, or is processed to provide,red blood cells suitable for use in humans, mammals, and/or vertebrates,such as for infusion. Red blood cell compositions include, for example,whole blood and red blood cell concentrates, such as packed red bloodcells (pRBCs; e.g., red blood cells with increased hematocrit and/or notcontaining additive solution). The red blood cell compositions may bedescribed by their hematocrit or packed cell volume (PCV), a measure ofthe concentration of red blood cells in the composition. Red blood cellcompositions may have a hematocrit in the range of about 1 to 100%, morelikely about 10 to 90%, also about 35 to 80%, or about 40 to 70%. Suchred blood cell compositions may include chemicals, such aspathogen-inactivating compounds and quenchers. They may also includebuffers and other solutions, such as red blood cell additive solutions(e.g., any solution described in herein, such as SAG-M, AS-5 or anysolution of Tables 2, 3, or 4), including salts or buffered solutions.In some embodiments, the red blood cell compositions described hereinare packed red blood cells having a hematocrit in the range of about 70to 90%, or about 75 to 85%, or about 80%, prior to use in the methods oftreating described herein. In some embodiments, the red blood cellcompositions are non-packed red blood cells having a hematocrit in therange about 50 to 70%, or about 55 to 65%, or about 60%, prior to and/orduring use in the methods of treating described herein. In someembodiments, the red blood cell compositions are diluted with a diluentsolution and have a hematocrit in the range about 30 to 50%, or about 35to 45%, or about 40%, prior to and/or during use in the methods oftreating described herein. In some embodiments, the red blood cellcompositions described herein have been leukoreduced prior to use in themethods of treating described herein. In some embodiments, the red bloodcell compositions have not been leukoreduced. Any red blood cellcomposition that will come into contact with, or be introduced into, aliving human, mammal, or vertebrate, where such contact carries a riskof transmitting disease due to contaminating pathogens may be treated asdisclosed herein.

Blood Pathogens

A pathogen contaminant, if present, to be inactivated in the methods ofthe invention includes any nucleic acid-containing agent capable ofcausing disease in a human, other mammals, or vertebrates. Thepathogenic agent may be unicellular or multicellular. Examples ofpathogens are bacteria, viruses, protozoa, fungi, yeasts, molds, andmycoplasmas which cause disease in humans, other mammals, orvertebrates. The genetic material of the pathogen may be DNA or RNA, andthe genetic material may be present as single-stranded ordouble-stranded nucleic acid. Table 1 lists examples of viruses, and isnot intended to limit the invention in any manner.

TABLE 1 Non-limiting examples of viruses Family: Virus: Adeno Adenovirus2 Canine hepatitis Arena Pichinde Lassa Bunya Turlock Californiaencephalitis Herpes Herpes simplex 1 Herpes simplex 2 CytomegalovirusPseudorabies Orothomyxo Influenza Papova SV-40 Paramyxo Measles MumpsParainfluenza 2 and 3 Picorna Poliovirus 1 and 2 Coxsackie A-9 Echo 11Pox Vaccinia Fowl Pox Reo Blue tongue Colorado tick fever Retro HIVAvian sarcoma Murine sarcoma Murine leukemia Rhabdo Vesicular stomatitisvirus Toga Western equine encephalitis Dengue 2 Dengue 4 St. Louisencephalitis Hepadna hepatitis B Bacteriophage Lambda R17 T2(Rickettsia) R. akari (rickettsialpox)

In addition to inactivating possible pathogen contaminants, the methodsof the present invention may also inactivate leukocytes that may bepresent in the red blood cell composition. Leukoreduction methods areused to preferably remove most of the leukocytes from red blood cellcompositions intended for infusion, as they may result in unwantedimmune responses in the recipient. However, not all blood isleukoreduced, or leukoreduction methods may not remove all of theleukocytes. Therefore, inactivation of any residual leukocytes by themethods of the invention as described herein may further reduce the riskof such immune responses.

Pathogen-Inactivating Compounds

The inactivation of a pathogen in the red blood cell compositions iseffected by contacting the pathogen in the red blood cell compositionwith a pathogen-inactivating compound. In any of the embodimentsdescribed herein, the pathogen-inactivating compound (e.g., S-303described herein) may be present in an effective amount (e.g., aneffective amount to inactivate a pathogen, such as an amount sufficientto inactivate, for example, at least 1 log, 2 log, or 3 log of apathogen in the red blood cell composition, if present).Pathogen-inactivating compounds that may be used by the methods of theinvention include compounds that comprise a functional group which is,or which is capable of forming and has formed, e.g. in situ, a reactivegroup, such as an electrophilic group. In some cases, thepathogen-inactivating compounds of the present invention do not requirephotoactivation to be reactive. For example, the functional group may bea mustard group, a mustard group intermediate, a mustard groupequivalent, an epoxide, a formaldehyde or a formaldehyde synthon. Suchfunctional groups are capable of forming in situ a reactive group, suchas an electrophilic aziridine, aziridinium, thiirane or thiiranium ion.A mustard group may be a mono- or bis-(haloethyl)amine group or amono(haloethyl)sulfide group. A mustard equivalent is a group thatreacts by a mechanism similar to the mustards, for example by formingreactive intermediates such as aziridinium and aziridine groups orthiirane and thiiranium groups. Examples include aziridine derivatives,mono or bis-(mesylethyl)amine groups, mono-(mesylethyl)sulfide groups,mono or bis-(tosylethyl)amine groups and mono-(tosylethyl)sulfidegroups. A formaldehyde synthon is any compound that breaks down to aformaldehyde, which includes a hydroxylamine such ashydroxymethylglycine. The reactive group of the pathogen-inactivatingcompound is capable of reacting with the nucleic acids of pathogens, forexample with nucleophilic groups on the nucleic acid. The reactive groupis also capable of reacting with a nucleophilic group of a quencher.Pathogen-inactivating compounds may also include a component thattargets the compound to nucleic acids, such as an anchor portion. Theanchor portion comprises a moiety which is capable of bindingnon-covalently to a nucleic acid biopolymer, such as DNA or RNA, and isalso referred to as a nucleic acid binding ligand, nucleic acid bindinggroup, or nucleic acid binding moiety. Examples of such compounds aredescribed in U.S. Pat. Nos. 5,691,132, 6,410,219, 6,136,586, 6,617,157,and 6,709,810, each of which is incorporated by reference herein.Another class of pathogen-inactivating compounds that may be quenched bythe methods of the invention comprises the above-mentioned reactivegroups linked to a nucleic acid binding group via a hydrolysable linker,as described in U.S. Pat. No. 6,514,987, incorporated by referenceherein. The anchor portion of the pathogen-inactivating compounds has anaffinity for nucleic acids. This affinity may be due to any of severalmodes of binding to the nucleic acid non-covalently, including, but notlimited to, intercalation, minor groove binding, major groove binding,and electrostatic binding (e.g., phosphate backbone binding). Theaffinity may also be due to mixed modes of binding (e.g., intercalationand minor groove binding). The binding may be sequence-specific (i.e.,increased binding affinity for one or more particular nucleic acidsequences over other nucleic acid sequences) or non sequence-specific.Detailed examples of such nucleic acid binding moieties can be found inthe above-mentioned patents.

In some embodiments of each of the methods, compositions, and kitsdescribed herein, the pathogen-inactivating compound may comprise afunctional group which is, or which forms, a reactive electrophilicgroup reactive with the nucleophile of the chosen quencher. In someembodiments, the pathogen-inactivating group comprises a nucleic acidbinding ligand and a functional group which is, or which forms anelectrophilic group.

A specific example of a suitable pathogen-inactivating compound for usein the present invention is β-alanine, N-(acridin-9-yl),2-[bis(2-chloroethyl)amino]ethyl ester (also alternatively referred toherein as “S-303”), the structure of which is as follows, includingsalts thereof.

In some embodiments, the concentration of the pathogen-inactivatingcompound, such as S-303, in the mixture with the red blood cellcomposition and the quencher is in the range of about 0.05 mM to 4 mM,about 0.05 mM to 2 mM, about 0.05 mM to 0.5 mM, about 0.1 mM to 0.3 mM,or about 0.2 mM. In some embodiments, the molar ratio of quencher topathogen inactivation compound once both components have been mixed withthe red blood cell composition is about 10:1 to about 400:1, also about10:1 to about 200:1, also about 20:1 to about 200:1, also about 50:1 toabout 200:1, also about 100:1.

Quenchers

Quenchers for use in methods of the present invention are intended toreduce unwanted side-reactions of the reactive electrophilic speciesused to inactivate pathogens (e.g., binding of the pathogen-inactivatingcompound to the RBC surface which may lead to an undesired immuneresponse). In any of the embodiments described herein, the quencher(e.g., glutathione described herein) may be present in an effectiveamount (for example, an effective amount to reduce unwanted sidereactions, such as the amounts described herein). Suitable quencherscomprise a nucleophilic group that is capable of reacting with theelectrophilic group of the pathogen-inactivating compound. Non-limitingexamples are described in detail in U.S. Pat. No. 6,709,810,incorporated by reference herein in its entirety. In some embodiments,the quenchers are capable of significantly reducing the unwanted sidereactions in a red blood cell composition while allowing thepathogen-inactivating compound to sufficiently inactivate a pathogenthat may be contaminating the red blood cell composition. In someembodiments, the improved methods of the present invention provide aneffective amount of quencher in combination with an effective amount ofpathogen-inactivating compound under conditions which provide optimalreduction in unwanted side reactions combined (e.g., binding of thepathogen-inactivating compound) with sufficient inactivation ofpathogens, without significantly altering (e.g., without decreasing) thecell osmotic fragility and without significantly altering (e.g., withoutincreasing) dehydration. A variety of unwanted side reactions may bereduced, such as reaction of the pathogen-inactivating compound withproteins and/or red blood cell components. In some embodiments, thequencher provides optimal reduction in the modification of the red bloodcells, such as the binding of IgG to the red blood cells or binding ofthe pathogen-inactivating compound to the red blood cells. While themethods of the invention involve the ex vivo treatment of red blood cellcompositions, some quenchers may remain in the composition uponintroduction into an individual. As such, in some embodiments, thequenchers of the invention are suitable for infusion. Suitable quenchersinclude, but are not limited to, compounds comprising a thiol group,such as quenchers comprising the amino acid cysteine or a suitablederivative of cysteine, such as N-acetyl cysteine. Examples of suchquenchers include cysteine and peptides comprising at least onecysteine, such as glutathione. In some embodiments, the suitablequenchers comprise a derivative of cysteine that can form a thiol groupin situ, with or without the use of additional chemicals or addedenzymes, such as S-acetyl cysteine or other suitable thiol derivedprodrugs of cysteine, or peptides comprising S-acetyl cysteine or othersuitable thiol derived prodrugs of cysteine. Suitable derivatives ofcysteine are those which either comprise, or are capable of forming insitu, a cysteinyl thiol which is capable of reacting with theelectrophilic group of the pathogen-inactivating compound.

In some embodiments, the quencher is a peptide of 2 to 10 amino acids,wherein at least one of the amino acids is cysteine, N-acetyl cysteine,S-acetyl cysteine, or other suitable derivative of cysteine. In someembodiments, the quencher is a peptide of at least 3 amino acids, suchas about 3-10 amino acids, also about 3-6 amino acids, wherein at leastone of the amino acids is cysteine, N-acetyl cysteine, S-acetylcysteine, or other suitable derivative of cysteine. In some embodiments,the quencher is a peptide of at least 3 amino acids, such as about 3-10amino acids, also about 3-6 amino acids, wherein at least one of theamino acids is cysteine, N-acetyl cysteine, S-acetyl cysteine, or othersuitable derivative of cysteine, also wherein at least 2 or at least 3of the amino acids is cysteine, N-acetyl cysteine, S-acetyl cysteine, orother suitable derivative of cysteine.

In a preferred embodiment, the quencher is neutralized glutathione (alsoknown as L-glutathione and γ-L-glutamyl-L-cysteinyl-glycine).Glutathione has many properties that make it particularly useful as aquencher. It is normally present in all cell types. It is not believedto be able to passively penetrate into a pathogen, such as by passingthrough cell membranes or lipid coats, of bacteria and lipid-envelopedviruses, or by passing through the viral capsid of non-envelopedviruses. At approximately neutral pH glutathione is charged and in theabsence of active transport, does not penetrate lipid bilayers to anysignificant extent. This is consistent with inactivation of lipidenveloped viruses such as HIV and VSV being substantially unaffected byglutathione, including using concentrations of neutralized glutathionegreater than 2 mM. The use of glutathione does have some effect oninactivation of e.g., Yersinia enterocolitica, Staphylococcusepidermidis and Serratia marcescens. However, this can be managed byusing effective amounts of neutralized glutathione andpathogen-inactivating compound. As such, preferred methods of quenchingare provided wherein contamination of a red blood cell composition by aviral or bacterial pathogen is inactivated by at least 2 log, preferablyat least 3 log. In some embodiments, Staphylococcus epidermidis may beinactivated by up to at least 3 log, also about 4 log, or about 5 logand VSV can be inactivated by up to at least 4 log, also about 5 log, orabout 6 log. In some embodiments, the inactivation of Staphylococcusepidermidis with S-303 is within about 3 log, also about 2 log, or about1 log that of a similar composition inactivated with 2 mM acidicglutathione and 0.2 mM S-303. In some embodiments, the inactivation ofVSV with S-303 is within about 2 log, or about 1 log, or essentiallyequal to that of a similar composition inactivated with 2 mM acidicglutathione and 0.2 mM S-303. At the appropriate conditions, asdescribed by the present invention, glutathione is also compatible within vitro storage of red blood cells and the resulting red blood cellcomposition is suitable for introduction in vivo.

In some embodiments, the quencher is glutathione in its reduced form.Glutathione disulfide, the oxidized form of glutathione, may also beused, so long as the glutathione disulfide is sufficiently reduced insolution prior to addition of the solution to the mixture comprising thered blood cell composition or sufficiently reduced after addition to themixture comprising the red blood cell composition.

In some embodiments, the quencher is a derivative of glutathione, suchas a glutathione monoalkyl ester or dialkyl ester, wherein the alkylgroup is a straight or branched group having 1 to 10 carbon atoms.Specific examples of alkyl groups include, but are not limited to methylgroup, ethyl group, propyl group, isopropyl group, butyl group, isobutylgroup, tert-butyl group, pentyl group, isopentyl group, neopentyl group,tert-pentyl group, 1-methylbutyl group, hexyl group, isohexyl group,2-methylpentyl group, 1-ethylbutyl group, heptyl group, octyl group,nonyl group, and decyl group. For instance, non-limiting examples ofglutathione derivatives include glutathione methyl ester, glutathionemonoethyl ester, and glutathione monoisopropyl ester. In someembodiments, glutathione oxidized diethyl ester(GSSG-(glycyl)-diethyl-ester) is used. In some embodiments, a thioesterof glutathione is hydrolyzed after addition to the red blood cellcompositions to form the thiol.

It is understood that in some embodiments, the quencher will be providedin the form of a free acid or base, whereas, in other embodiments, thequencher will be provided in the form of a salt. If the quencher is inthe form of a salt, the salt is preferably a pharmaceutically acceptablesalt. The pharmaceutically-acceptable salts of compounds (in the form ofwater- or oil-soluble or dispersible products) include the conventionalnon-toxic salts or the quaternary ammonium salts which are formed, e.g.,from inorganic or organic acids or bases. Examples of such acid additionsalts include acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-napthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.Base salts include ammonium salts, alkali metal salts such as sodium andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth. Also, the basic nitrogen-containinggroups may be quaternized with such agents as lower alkyl halides, suchas methyl, ethyl, propyl, and butyl chloride, bromides and iodides;dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates,long chain halides such as decyl, lauryl, myristyl and stearylchlorides, bromides and iodides, aralkyl halides like benzyl andphenethyl bromides and others. Other pharmaceutically acceptable saltsinclude the sulfate salt ethanolate and sulfate salts.

For example, in some embodiments, the quencher is in the form of apharmaceutically acceptable salt formed from glutathione. In someembodiments, the quencher is in the form of a pharmaceuticallyacceptable salt formed from glutathione and one or more cations such assodium, aluminum, calcium, lithium, magnesium, zinc, ortetramethylammonium. In some embodiments, the quencher is glutathione(reduced) and is provided in the form of glutathione monosodium salt(available, e.g., from Biomedica Foscama, Italy). In some otherembodiments, the glutathione (reduced) is provided in the form ofglutathione hydrochloride salt. In some other embodiments, theglutathione is provided in the form of a glutathione (reduced) disodiumsalt. In further embodiments, a glutathione monoalkyl ester sulfate isused. In some embodiments, glutathione is provided in the form ofglutathione oxidized disodium salt.

Methods of Inactivation and Quenching

The methods of the present invention involve the combination of a redblood cell composition with a pathogen-inactivating compound and aquencher under conditions wherein, upon mixing the composition with thepathogen-inactivating compound and quencher, the pH of the resultingcomposition is in a suitable range to provide adequate pathogeninactivation and reduction of unwanted side reactions (such asmodification of the red blood cells) with limited or no effect on thevitality (e.g. osmotic fragility and dehydration) and/or lifespan of thetreated blood product. Further, the present invention describesdecreasing the concentration of the quencher in the red blood cellcomposition following a period of pathogen inactivation to aid inmaintaining the vitality and lifespan of the red blood cells duringstorage. An additive solution, as described herein, also may be utilizedfor the red blood cells during storage and may be used to replacetreatment solutions and/or diluent solutions used during pathogeninactivation.

The improved methods include several features that may be important forquenching. The first feature is the thiol group, or other suitablenucleophilic group. The second is the adjustment of the pH of thesolution. It is possible to provide some level of quenching by suitablyadjusting the pH of the solution. As such, the quenchers of theinvention provide some buffering capacity to the composition comprisingred blood cells, where the buffering capacity itself provides improvedquenching. For example, using a cysteine analog such as methionine as aquencher, when appropriately modified to provide a suitable pH change inthe red blood cell composition, will result in some level of quenchingof binding of the pathogen-inactivating compound to the red blood cells.As the sulfur atom in methionine is not nucleophilic, methionine doesnot provide any quenching other than providing the necessary pH of thesolution. Thus, the combination of pH adjustment and a thiol groupprovides improved quenching. Proper adjustment of pH and base equivalentmay also decrease the level of red blood cell dehydration during theinactivation period. A third feature that may be important for providingimproved quenching in some embodiments, is selection of preferredquenchers that do not substantially penetrate inside of pathogens suchas viruses and bacteria. Such quenchers provide adequate quenching inthe extracellular environment, where detrimental reactions such asbinding to red cell surfaces occur, without additional quenching ofpathogen-inactivating compound once it has penetrated inside of thepathogen. Finally, the improved quenching methods of the presentinvention include decreasing the quencher concentration followinginactivation and, in some cases, adding an additive solution forstorage. The red blood cells have been shown to have improved lifespanand decreased levels of dehydration during storage when the overallconcentration of quencher is decreased to suitable levels.

In one aspect, the present invention provides a method of treating a redblood cell composition comprising: a) providing i) apathogen-inactivating compound comprising a functional group which is,or which forms, a reactive electrophilic group (e.g., an effectiveamount of a pathogen-inactivating compound to inactivate a pathogen, ifpresent), ii) a quencher (e.g., an effective amount of a quencher asdescribed herein) comprising a thiol group, wherein the thiol is capableof reacting with the reactive electrophilic group of thepathogen-inactivating compound, and iii) a composition comprising redblood cells; b) mixing the pathogen-inactivating compound and quencherwith the composition comprising red blood cells; and c) sufficientlydecreasing the concentration of the quencher in the mixture to an amountwhich reduces the level of red blood cell dehydration resulting fromstorage of the mixture, relative to the level of red blood celldehydration resulting from storage of the mixture at the originalconcentration of quencher. In some embodiments, step (a) furthercomprises providing a suitable base, step (b) further comprises mixingthe base with the composition comprising red blood cells, and the baseis of sufficient amount to reduce the level of an unwanted reaction ofthe pathogen-inactivating compound with red blood cells in the mixture,relative to the mixture without the base. In some embodiments, theunwanted reaction of the pathogen-inactivating compound with red bloodcells is modification of the surface of the red blood cells by thepathogen-inactivating compound. In some embodiments, step (a) furthercomprises providing a suitable base, step (b) further comprises mixingthe base with the composition comprising red blood cells, and the baseis of sufficient amount to reduce the level of anti-pathogeninactivating compound antibody binding to the treated red blood cellcomposition in the resulting mixture by at least about 5% (or at leastabout 10%, at least about 25%, at least about 50%, at least about 75%,or at least about 90%) relative to the mixture without the base. In someembodiments, storage of the mixture is greater than, equal to, or lessthan 7, 10, 14, 21, 28, 35, or 42 days of storage at 4° C. or roomtemperature. In some embodiments, the mixture is stored in an additivesolution (e.g., any additive solution described in Table 2, and/or anadditive solution comprising sodium chloride, adenine, glucose,phosphate, guanosine, citrate and/or mannitol). In some embodiments, themethod further comprises replacing the solution used during treatmentwith an additive solution (e.g., any additive solution described inTable 2, and/or an additive solution comprising sodium chloride,adenine, glucose, phosphate, guanosine, citrate and/or mannitol).

In an additional aspect, the invention provides a method of reducingdehydration in red blood cells, comprising: a) providing a red bloodcell composition comprising i) a quencher, where the quencher is capableof reacting with a pathogen-inactivating compound, and ii) red bloodcells; and b) sufficiently decreasing the concentration of the quencherin the mixture to an amount which reduces the level of red blood celldehydration resulting from storage of the mixture relative to the levelof red blood cell dehydration resulting from storage of the mixture atthe original concentration of quencher. In some embodiments, storage ofthe mixture is greater than, equal to, or less than 7, 10, 14, 21, 28,35, or 42 days of storage at 4° C. or room temperature. In someembodiments, the method further comprises the addition of an additivesolution (e.g., any additive solution described in Table 2, and/or anadditive solution comprising sodium chloride, adenine, glucose,phosphate, guanosine, citrate and/or mannitol), e.g., prior to storage.

The quencher and/or added base (or the neutralized quencher) used in themethods described herein may be mixed with the red blood cellcomposition prior to, at the same time as, or after addition of thepathogen-inactivating compound to the red blood cell composition. If thequencher and base (or neutralized quencher) are mixed with the red bloodcell composition after the pathogen-inactivating solution is mixed withthe red blood cell composition, the quencher and/or base (or neutralizedquencher) are preferably added to the red blood cell composition beforea significant amount of side reaction of the pathogen-inactivatingcompound with the red blood cells has occurred, so that adequatequenching of the undesired side reaction can be achieved. In someembodiments, the quencher and/or base (or neutralized quencher) is mixedwith the red blood cell composition within about an hour, within about30 minutes, within about 20 minutes, within about 10 minutes, withinabout 5 minutes, within about 2 minutes, or within about 1 minute aftermixing the pathogen-inactivating compound with the red blood cellcomposition. In some embodiments, the quencher and base are mixed withthe red blood cell composition at the same time as thepathogen-inactivating compound.

In some embodiments of each of the methods described herein, thequencher and the added base (or the neutralized quencher) are pretreatedwith the red blood cell composition for a suitable time interval priorto addition of the pathogen-inactivating compound (e.g., S-303), such asless than about an hour, less than about 30 minutes, less than about 20minutes, less than about 10 minutes, less than about 5 minutes, lessthan about 2 minutes, or less than about 1 minute before mixing thepathogen-inactivating compound with the red blood cell composition. Insome further embodiments, the pretreatment is at a temperature of about1° C. to 30° C., also about 18° C. to 25° C., or about 37° C., or aboutroom temperature.

In some embodiments of each of the methods described herein, thepathogen-inactivating compound (e.g., S-303) is incubated with the redblood cell composition in the presence of the quencher and the addedbase (or the neutralized quencher) for a suitable time interval, such asfor about 30 minutes to 48 hours, also about 2 to 36 hours, also about 8to 24 hours, also about 20 hours. In some further embodiments, theincubation is in a temperature range of about 1° C. to 30° C., alsoabout 18° C. to 25° C., or about 37° C., or about room temperature.

With respect to the feature of adjusting the pH of the red blood cellcomposition, the previous methods of quenching suchpathogen-inactivating compounds fail to recognize the importance of thepH of the resulting mixture with respect to both quenching effectivenessand cell vitality during inactivation. While the previous methods havedemonstrated the need for sufficient base and suitable pH level foradequately quenching unwanted side reactions of thepathogen-inactivating compound (e.g., by increasing the levels ofnon-protonated glutathione to reduce binding of thepathogen-inactivating compound to the RBC surface), these methods do notrealize and describe the effects of the increased base on celldehydration during the inactivation process. Thus, one aspect of thepresent invention involves adjusting the pH of the red blood cellcomposition to a suitable level for the incubation of thepathogen-inactivating compound and quencher (e.g., a suitable level toavoid adversely affecting dehydration).

In some embodiments, upon mixing the pathogen-inactivating compound andquencher with the red blood cell composition, the pH of the mixture isat suitable level to reduce unwanted side reactions of thepathogen-inactivating compound (e.g., binding of thepathogen-inactivating compound to the RBC surface which may lead to anundesired immune response) and sufficiently reduce cell dehydrationduring inactivation. In some embodiments, the unwanted side reaction ismodification of the surface of the red blood cells by thepathogen-inactivating compound. In some embodiments, the modification iscovalent binding of the pathogen-inactivating compound to the surface ofthe red blood cells. In other embodiments, the modification isnon-covalent binding of the pathogen-inactivating compound to thesurface of the red blood cells.

As described herein, in some embodiments of each of the methods, anundesired (also referred to herein as “unwanted”) side reaction of thepathogen inactivating compound with the red blood cells is reduced. Insome embodiments, the undesired side reaction that is reduced ismodification of the red blood cell surface by the pathogen inactivatingcompound. In some embodiments, the level of side reaction is reduced byat least about 5%, at least about 10%, at least about 25%, at leastabout 50%, at least about 75%, or at least about 90%. The decrease inthe side reaction may be evidenced, for example, by measuring the amountof binding to the treated red blood cells of antibodies specific to thepathogen inactivating compound and/or measuring the life span of thetreated red blood cells in vivo, and comparing these measurements to redblood cells treated by a second, different method (for example, a methodwithout sufficient quencher and/or base added to the reaction mixture, amethod in which no quencher and/or base is added to the reactionmixture, and/or a treatment at a lower pH). For instance, in someembodiments of the methods described herein, the level of anti-pathogeninactivating compound antibody binding to the treated blood cells isdecreased by at least about 10%%, at least about 25%, at least about50%, at least about 75%%, or at least about 90%, relative to a secondmethod (e.g., a method without sufficient quencher and/or base added tothe reaction mixture, a method in which no quencher and/or base is addedto the reaction mixture, and/or treatment at a lower pH).

In some embodiments, upon mixing the pathogen-inactivating compound andquencher with the red blood cell composition, the pH of the mixture isin the range of about 6.0 to 8.5, about 6.0 to 7.5, about 6.5 to 7.1,about 6.5 to 7.0, about 6.6 to 6.8, or about 6.6, 6.7, 6.8, or 6.9.While the pH in a red blood cell composition may change with time, it isdesirable that the pH is in a desired range when quencher is added tothe red blood cell composition, whether or not it already containspathogen-inactivating compound. The methods of the present inventioninvolve adding pathogen-inactivating compound and quencher to a redblood cell composition. The desired pH range is necessary upon theaddition of both the pathogen-inactivating compound and quencher,regardless of the order of addition of the pathogen-inactivatingcompound and/or quencher to the red blood cell composition. In otherwords, once all three components have been mixed, the pH is within thedesired range. In some embodiments, quencher is added prior topathogen-inactivating compound. In some embodiments,pathogen-inactivating compound is added prior to quencher. In someembodiments, quencher and pathogen-inactivating compound are addedessentially simultaneously. Thus, upon addition of pathogen-inactivatingcompound and quencher means at the point when both of the quencher andpathogen-inactivating compound have been mixed with the red blood cellcomposition. The desired pH can be achieved by several means, and is notlimited as to when the pH of the red blood cell composition is adjusted,or in some embodiments, is not significantly adjusted from the naturalpH of the blood product. For example, the desired pH of the red bloodcell composition can be achieved by adjusting the pH. The pH adjustmentmay be done, for example, by addition of a suitable additive solution,such as a buffering solution, prior to adding the pathogen-inactivatingcompound and quencher. In some embodiments, the red blood cellcomposition may be washed one or more times with a suitable bufferbefore suspending in the same or other suitable buffer. Alternatively,the pH of the red blood cell composition can be adjusted simultaneouslywith the addition of either the pathogen-inactivating compound, thequencher, or both. In some embodiments, the pH is adjustedsimultaneously with addition of the quencher. In some embodiments, thequencher is neutralized, such that addition of the neutralized quencherprovides the desired pH range in the red blood cell composition. As anexample, the neutralization of glutathione can be used to effect thenecessary pH adjustments. In some embodiments, an appropriate level ofneutralization of the glutathione can be used, for example by additionof 1 equivalent of base, to provide a quencher that, upon addition to ared blood cell composition, will provide the necessary pH adjustment ofthe composition. The appropriate neutralization will depend upon thequencher used. For example, when a peptide is used it may depend on theamino acid components of the peptide. In some embodiments, a quenchercan be used that does not significantly affect the pH of the red bloodcell composition. For example, use of a peptide comprising a cysteinethat may further comprise one or more amino acids that result in a moreneutral pH for a solution of the naturally isolated peptide. In someembodiments, the peptide further comprises at least one basic aminoacid, such as arginine or lysine.

In some embodiments of the methods described herein, where a base ismixed with the red blood cell composition along with thepathogen-inactivating compound and quencher to increase the pH of themixture to a desired level and/or to improve quenching of undesired sidereactions, the base is a basic salt. The basic salt may first bedissolved in an aqueous solution prior to mixing with the red blood cellcomposition. In other embodiments, the salt may be added directly to thered blood cell composition in solid form. In some embodiments, the basicsalt comprises the quencher and provides both the quencher and the baseto the mixture. In some embodiments, the base used in the method is astrong base, such as NaOH. Typically, a strong base like NaOH will bedissolved first in aqueous solution prior to mixing with the red bloodcell composition. In some embodiments, the strong base (e.g., insolution or solid form) is mixed with the quencher prior to mixing thequencher with the red blood cell composition. In some embodiments, thebase is a basic buffer (added in sufficient quantities and having anappropriate pKa to bring the mixture to the desired pH range). If abasic buffer is used, the buffer will, in some embodiments, be apharmaceutically acceptable buffer. In some embodiments, the buffer willhave a titratable proton with a pKa in the range of about 7 to 8.Examples of buffers which can be used as basic buffers include, but arenot limited to, N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid(HEPES), phosphate buffered saline (PBS), and sodium phosphate buffer.Other suitable basic buffers will be readily identifiable by one ofordinary skill in the art.

In some embodiments of each of the methods and compositions describedherein, the pH of the mixture of red blood cells, quencher,pathogen-inactivating compound, and any added base is greater than about5.5, greater than about 5.7, greater than about 6.0, greater than about6.3, greater than about 6.5, greater than about 6.7, greater than about7.0, or greater than about 7.2. In some embodiments of each of themethods and compositions described herein, the pH of the mixture of redblood cells, quencher, pathogen-inactivating compound, and base (if anyis added) is in the range of about 6.0 to 8.5, about 6.0 to 7.5, about6.5 to 7.1, about 6.5 to 7.0, or about 6.6 to 6.8, or about 6.6, 6.7,6.8, or 6.9. In some embodiments, the indicated pH is the pH at roomtemperature. In some embodiments, the indicated pH is the pH at 37° C.For example, in some embodiments, the composition comprising the redblood cells are treated with the pathogen-inactivating compound in thepresence of the quencher and any added base, wherein the pH of themixture is in the range of about 6.5 to about 7.0 (or 7.1) at 37° C.

In some embodiments, the pH of the mixture of red blood cells, quencher,and the base (if base is added as part of the method) is in the range ofabout 6.0 to 8.5, about 6.0 to 7.5, about 6.5 to 7.1, about 6.5 to 7.0,or about 6.6 to 6.8, or about 6.5, 6.7, 6.8, or 6.9, prior to mixing thepathogen-inactivating compound with the red blood cell composition. Insome other embodiments, the pH is achieved at the same time as or withinabout 1 hour, within about 30 minutes, within about 20 minutes, withinabout 10 minutes, within about 5 minutes, or within about 2 minutes ofmixing the pathogen-inactivating compound with the compositioncomprising the red blood cells. In some embodiments of those methodswhere the pH is adjusted, the pH is adjusted to the desired pH rangeprior to, at the same time as, within about 1 hour, within about 30minutes, within about 20 minutes, within about 10 minutes, within about5 minutes, or within about 2 minutes of mixing the pathogen-inactivatingcompound with the composition comprising the red blood cells. In thoseembodiments, where the quencher is glutathione and thepathogen-inactivating compound is S-303, the pH of the mixturecomprising the red blood cell composition and the quencher is preferablyadjusted to the desired pH range (e.g., pH 6.5 to 7.0) prior to mixingthe S-303 with the red blood cell composition.

In some embodiments, the resulting pH of the composition after mixingthe red blood cells, quencher, and the base, is not necessarily anadjustment of the pH of the starting red blood cell composition. Forexample, a red blood cell composition may have a pH in the desired rangeof 6.0-7.5, and the pH of the composition does not change significantlyon addition of quencher, and subsequently pathogen-inactivatingcompound. In such embodiments, the quencher either naturally providesthe desired pH, or is neutralized accordingly to provide the desired pH.It is the combination of adding high initial amounts of quencher, suchas about 5 mM to about 40 mM, with a resulting pH in the desired rangethat is important. Known methods using such concentrations ofglutathione, for example, have not been used with the desired pH rangein conjunction with other aspects of the present invention. Thus, forpeptides, regardless of the peptide quencher, it can be effectivelyneutralized as needed to provide a suitable pH range when added to a redblood cell composition, and further may be selected to provide asuitable amount of buffering in the desired pH range. As such, aneutralized quencher means that the quencher is suitably titrated withacid or base as needed such that on addition to a red blood cellcomposition, the resulting mixture has a pH that provides betterquenching of unwanted side reactions (e.g., binding of thepathogen-inactivating compound to the RBC surface which may lead to anundesired immune response) while avoiding cell dehydration duringinactivation, such as a pH in the range of about 6.0 to 8.5, about 6.0to 7.5, about 6.5 to 7.0, about 6.5 to 7.1, or about 6.6 to 6.8, orabout 6.6, 6.7, 6.8, or 6.9. In some embodiments, the peptide asisolated naturally, is suitably neutralized, i.e. requires no additionof acid or base to provide the desired pH in the final mixture. Further,preferred quenchers will provide buffering capacity to maintain the pHin the desired range for a time necessary to quench unwanted sidereactions.

In some embodiments of each of the methods and compositions describedherein, the quencher is neutralized. A quencher is said to be“neutralized” by a base, if a sufficient amount of the base has beencombined with the quencher, such that the quenching of an undesired sidereaction between the pathogen-inactivating compound and the red bloodcells is improved in a mixture comprising the composition comprising thered blood cells, the pathogen-inactivating compound, and quencher. A“neutralized quencher” does not necessarily have a neutral pH, nor is itnecessarily uncharged. In some embodiments, the neutralized quencher isneither in its most protonated form nor its most deprotonated form. Insome embodiments, where the quencher is very acidic, the pH of theneutralized quencher may still be lower than 7.0 (e.g., about 6.6, 6.7,6.8, or 6.9). In some embodiments, the pH of solution of the neutralizedquencher may be greater than 7.0. In some embodiments, the pH of thesolution of the neutralized quencher will be detectably higher than thatof the quencher prior to addition of the base. In some embodiments, thequencher is neutralized with at least about 0.25 equivalents, at leastabout 0.5 equivalents, at least about 0.75 equivalents, at least about 1equivalent, at least about 1.25 equivalents, at least about 1.5equivalents, or at least about 2 equivalents of a base. In someembodiments, the quencher is neutralized with less than about 2equivalents, less than about 1.5 equivalents, less than about 1.25equivalents, less than about 1 equivalent, or less than about 0.75equivalents of a base. In some embodiments, the quencher is neutralizedwith about 0.25 to about 2 equivalents, about 0.5 to about 1.5equivalents, or about 0.75 to about 1.25 equivalents of base. In someembodiments, the quencher is neutralized with about 0.75 equivalent ofbase. In other embodiments, the quencher is neutralized with about 1equivalent of base. In other embodiments, the quencher is neutralizedwith about 1.25 equivalent of base. For example, In some embodiments ofthe invention, glutathione is neutralized with about 1 equivalent of asuitable base, such as sodium hydroxide. In this instance, a solution ofthe protonated glutathione has a pH of approximately 3, a solutionneutralized with 1 equivalent of sodium hydroxide has a pH ofapproximately 4.5, and a solution neutralized with 2 equivalents ofsodium hydroxide has a pH of approximately 9.5. Any appropriate peptidequencher comprising at least one cysteine can be suitably adjusted toprovide the desired pH upon addition to the red blood cell composition.

Appropriate methods for neutralizing glutathione and other quencherswill be readily apparent to those of ordinary skill in the art. In someembodiments, sodium hydroxide is used to neutralize or partiallyneutralize the quencher. In some embodiments, solid pellets of NaOH arefirst dissolved in water to generate a concentrated solution of thebase, such as a 1 N, 5 N, 10 N, or 20 N NaOH solution. In someembodiments, an appropriate amount of that NaOH solution is then addedto the quencher either prior to, at the same time as, or followingaddition of the quencher to the mixture. Alternatively, the NaOH isadded to the red blood cell composition or the pathogen-inactivatingcompound, or the combination of the two, prior to the addition of thequencher to the mixture.

In addition to providing a quencher that is suitably pH-adjusted orneutralized, in some embodiments, preferred quenchers are not able tosignificantly enter into the pathogens, such that they optimally quenchunwanted reactions in the extracellular environment, but do notinterfere with pathogen inactivation once the pathogen-inactivatingcompound has penetrated inside of the pathogen.

In some embodiments of each of the methods described herein, thequencher is an acidic compound. In some embodiments, the quencher isprovided in the free acid form. In some embodiments, the quencher isacidic and at least about 1 equivalent of base is added to neutralizethe quencher. A solution comprising such a neutralized quencher may be,in some instances, basic, neutral, or even acidic. In some embodiments,about 1 equivalent of base is added to neutralize or partiallyneutralize the quencher. In some embodiments, about 2 equivalents ofbase are added. In some embodiments, the quencher is acidic and about0.5 to about 1.5 equivalents of base is used to neutralize the quencher.In some embodiments, about 0.75 to about 1.25 equivalents of base areused. In some embodiments, about 1 equivalent of base is used.

In some embodiments, the quencher is neutralized prior to addition tothe red blood cell composition and/or pathogen-inactivating compound. Inother embodiments, the quencher is neutralized after combining thequencher with either the red blood cell composition and/orpathogen-inactivating compound. In some embodiments, the pH of theneutralized quencher prior to addition to the red blood cell compositionand/or pathogen-inactivating compound is in the range of about 2.5 to7.5, about 3.0 to 6.5, about 3.5 to 5.5, about 4.0 to 5.0, or about 4.3to 4.5, or about 4.4.

In some embodiments, the quencher is glutathione and is provided in theform of glutathione monosodium salt and is neutralized with about 1equivalent of base, or is not neutralized with base. In some otherembodiments, the quencher is glutathione and is provided in the form ofglutathione hydrochloride salt and is neutralized with about 1equivalent of base.

In some embodiments of each of the methods described herein, the initialconcentration of the quencher in the mixture comprising the red bloodcell composition, quencher, pathogen-inactivating compound, and anyadded base is elevated during a period for inactivation, and thenreduced to a lowered concentration following the period of inactivation.In some embodiments, the initial concentration of the quencher isadequate to sufficiently reduce unwanted side reactions of thepathogen-inactivating compound (e.g., binding of thepathogen-inactivating compound to the RBC surface), then reduced to alowered concentration to sufficiently reduce adversely affecting thevitality (e.g., osmotic fragility and dehydration) and/or lifespanduring cell storage.

The invention embraces any number of methods used to reduce theconcentration of quencher following the period of pathogen inactivation.In some embodiments, the concentration of quencher (e.g., glutathione)is reduced by centrifugation of the mixture comprising the red bloodcell composition, quencher, and pathogen-inactivating compound, followedby removal of the supernatant of the mixture, then the addition of freshsolution, such as an additive solution (e.g., any additive solutiondescribed in Table 2, and/or an additive solution comprising sodiumchloride, adenine, glucose, phosphate, guanosine, citrate and/ormannitol), for resuspension of the cells (e.g., via washing the cells).The process of centrifugation, supernatant removal, and addition offresh solution (e.g., any additive solution described in Table 2, and/oran additive solution comprising sodium chloride, adenine, glucose,phosphate, guanosine, citrate and/or mannitol), may be, in someembodiments, repeated for an additional 1, 2, 3, 4, or 5 or more times.In some embodiments, the method used to reduce the concentration of thequencher is automated. In some embodiments, the fresh solution does notcomprise the quencher or comprises a lower concentration of thequencher. In some embodiments, the concentration of quencher (e.g.,glutathione) is reduced by chemically deactivating the quencher. In someembodiments, the concentration of quencher (e.g., glutathione) isreduced by adsorption in a batch or flow removal process or sizeexclusion in flow process using membranes (e.g., hollow fiber membranesor dialysis membranes), or size exclusion heads. In some embodiments,the quencher is not reduced and/or is not contacted with a compoundadsorption device (CAD).

In some embodiments of each of the methods and compositions describedherein, the initial concentration of the quencher (e.g., glutathione) inthe mixture comprising the red blood cell composition, quencher,pathogen-inactivating compound, and any added base is greater than about2 mM, greater than about 4 mM, greater than about 6 mM, greater thanabout 8 mM, greater than about 10 mM, greater than about 15 mM, orgreater than about 20 mM. In some embodiments, the initial quencherconcentration in the mixture is in the range of about 2 mM to 100 mM,about 2 mM to 40 mM, about 4 mM to 40 mM, about 5 mM to 40 mM, about 5mM to 30 mM, or about 10 mM to 30 mM, or up to 2 mM, 5 mM, 10 mM, 15 mM,20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or 100 mM. In someembodiments, the initial quencher concentration in the mixture is about20 mM.

In some embodiments of each of the methods and compositions describedherein, the initial concentration of quencher (e.g., glutathione) in themixture of red blood cells, quencher, and the pathogen-inactivatingcompound is greater than about 2 mM, greater than about 4 mM, greaterthan about 6 mM, greater than about 8 mM or greater than about 10 mM,and the pH of the mixture is greater than about 5.5, greater than about5.7, greater than about 6.0, greater than about 6.3, greater than about6.5, greater than about 6.7, greater than about 7.0, or greater thanabout 7.2. In some embodiments of each of the methods and compositionsdescribed herein, the initial concentration of the quencher in themixture is in the range of about 2 mM to 40 mM, about 4 mM to 40 mM,about 5 mM to 40 mM, about 5 mM to 30 mM, or about 10 mM to 30 mM, orabout 20 mM, and the pH of the mixture is in the range of about 6.0 to8.5, about 6.0 to 7.5, about 6.5 to 7.1, about 6.5 to 7.0, or about 6.6to 6.8, or about 6.6, 6.7, 6.8, or 6.9. In some embodiments, the initialconcentration of quencher in the mixture is greater than about 2 mM,greater than about 4 mM, greater than about 6 mM, greater than about 8mM or greater than about 10 mM, and the pH of the mixture is in therange of about 6.0 to 8.5, about 6.0 to 7.5, about 6.5 to 7.1, about 6.5to 7.0, or about 6.6 to 6.8, or about 6.6, 6.7, 6.8, or 6.9. In someembodiments, the concentration of quencher (e.g., glutathione) in themixture is in the range of about 10 mM to about 30 mM, and the pH of themixture is in the range of about 6.0 to 7.5. In some embodiments, theconcentration of quencher (e.g., glutathione) in the mixture is in therange of about 20 mM, and the pH of the mixture is in the range of about6.5 to 7.0 (or 7.1).

In some embodiments of each of the methods and compositions describedherein, the initial concentration of the quencher (e.g., glutathione) inthe mixture comprising the red blood cell composition, quencher,pathogen-inactivating compound and any added base following the periodof inactivation is reduced by greater than 2-fold, or 3-fold, or 4-fold,or 5-fold, or 6-fold, or 7-fold, or 8-fold, or 9-fold, or 10-fold, or15-fold, or 20-fold, or 25-fold, or 30-fold, or 35-fold, or 40-fold, or50-fold, or 100-fold, or 500-fold, or 1000-fold relative to the initialconcentration of the quencher (e.g., glutathione) in the mixture.

In some embodiments of each of the methods and compositions describedherein, the lowered concentration of the quencher (e.g., glutathione) inthe mixture comprising the red blood cell composition, quencher,pathogen-inactivating compound and any added base following the periodof inactivation is less than about 15 mM, less than about 10 mM, lessthan about 8 mM, less than about 6 mM, less than about 5 mM, less thanabout 4 mM, less than about 3 mM, less than about 2 mM, less than about1 mM, less than about 0.75 mM, less than about 0.5 mM, or less thanabout 0.25 mM. In some embodiments, the lowered concentration of thequencher in the mixture following the period of inactivation is in therange of about 1 mM to 20 mM, about 2 mM to 15 mM, about 3 mM to 10 mM,about 4 mM to 8 mM, or about 5 mM to 6 mM. In some embodiments, thelowered concentration of the quencher in the mixture following theperiod of inactivation is at a concentration of up to about 0.25 mM, or0.5 mM, or 0.75 mM, or 1 mM, or 1.5 mM, or 2 mM, or 3 mM, or 4 mM, or 5mM, or 6 mM, or 7 mM, or 8 mM, or 9 mM, or 10 mM, or 12.5 mM, or 15 mM,or 20 mM.

In some embodiments of each of the methods and compositions describedherein, the initial concentration of the quencher (e.g., glutathione) inthe mixture comprising the red blood cell composition, quencher,pathogen-inactivating compound and any added base is greater than 2 mM,greater than about 4 mM, greater than about 6 mM, greater than about 8mM or greater than about 10 mM, and the lowered concentration of thequencher in the mixture following the period of inactivation is lessthan about 15 mM, less than about 10 mM, less than about 8 mM, less thanabout 6 mM, less than about 5 mM, less than about 4 mM, less than about3 mM, less than about 2 mM, less than about 1.5 mM, less than about 1mM, less than about 0.75 mM, less than about 0.5 mM, or less than about0.25 mM. In some embodiments, the initial concentration of the quencheris in the range of about 2 mM to 100 mM, about 2 mM to 40 mM, about 4 mMto 40 mM, about 5 mM to 40 mM, about 5 mM to 30 mM, or about 10 mM to 30mM, or about 20 mM, and the lowered concentration of the quencher in themixture following the period of inactivation is in the range of about 1mM to 20 mM, about 2 mM to 15 mM, about 3 mM to 10 mM, about 4 mM to 8mM, or about 5 mM to 6 mM. In some embodiments, the initialconcentration of the quencher (e.g., glutathione) is in the range ofabout 10 mM to 30 mM, and the lowered concentration of the quencher inthe mixture following the period of inactivation is in the range ofabout 2 mM to 15 mM. In some embodiments, the initial concentration ofthe quencher (e.g., glutathione) is about 20 mM, and the loweredconcentration of the quencher in the mixture following the period ofinactivation is in the range of about 4 mM to 8 mM.

In some embodiments, the initial concentration of the quencher (e.g.,glutathione) in the mixture comprising the red blood cell composition,quencher (e.g., glutathione), pathogen-inactivating compound and anyadded base is greater than 2 mM, greater than about 4 mM, greater thanabout 6 mM, greater than about 8 mM or greater than about 10 mM; the pHof the mixture is greater than about 5.5, greater than about 5.7,greater than about 6.0, greater than about 6.3, greater than about 6.5,greater than about 6.7, greater than about 7.0, or greater than about7.2; and the lowered concentration of the quencher in the mixturefollowing the period of inactivation is less than about 15 mM, less thanabout 10 mM, less than about 8 mM, less than about 6 mM, less than about5 mM, less than about 4 mM, less than about 3 mM, less than about 2 mM,less than about 1.5 mM, less than about 1 mM, less than about 0.75 mM,less than about 0.5 mM, or less than about 0.25 mM. In some embodiments,the initial concentration of the quencher is in the range of about 2 mMto 100 mM, about 2 mM to 40 mM, about 4 mM to 40 mM, about 5 mM to 40mM, about 5 mM to 30 mM, or about 10 mM to 30 mM, or about 20 mM; the pHof the mixture is in the range of about 6.0 to 8.5, about 6.0 to 7.5,about 6.5 to 7.0, about 6.5 to 7.1, or about 6.6 to 6.8, or about 6.6,6.7, 6.8, or 6.9; and the lowered concentration of the quencher in themixture following the period of inactivation is in the range of about 1mM to 20 mM, about 2 mM to 15 mM, about 3 mM to 10 mM, about 4 mM to 8mM, or about 5 mM to 6 mM. In some embodiments, the initialconcentration of the quencher (e.g., glutathione) is in the range ofabout 10 mM to 30 mM; the pH of the mixture is in the range of about 6.0to 7.5; and the lowered concentration of the quencher in the mixturefollowing the period of inactivation is in the range of about 2 mM to 15mM. In some embodiments, the initial concentration of the quencher(e.g., glutathione) is about 20 mM; the pH of the mixture is in therange of about 6.5 to 7.0 (or 7.1); and the lowered concentration of thequencher in the mixture following the period of inactivation is in therange of about 4 mM to 8 mM.

In some embodiments of each of the methods and compositions describedherein, the period of time between the point of addition of the quencherat the initial concentration and the point of reducing the concentrationof the quencher to a lowered concentration in the mixture comprising thered blood cell composition, quencher, pathogen-inactivating compound,and any added base is sufficient to reduce unwanted side reactions ofthe pathogen-inactivating compound (e.g., binding of thepathogen-inactivating compound to the RBC surface which may lead to anundesired immune response). In some embodiments, the period of time issufficient to reduce unwanted side reactions of thepathogen-inactivating compound and to avoid or reduce cell dehydrationduring the inactivation process.

In some embodiments, the period of time between the point of addition ofthe quencher at the initial concentration and the point of reducing theconcentration of the quencher to a lowered concentration in the mixturecomprising the red blood cell composition, quencher,pathogen-inactivating compound, and any added base is greater than,about equal to, or less than 5 hours, 10 hours, 15 hours, 20 hours, 25hours, 30 hours, 35 hours, 40 hours, or 50 hours. In some embodiments,the period of time is about 1 to 96 hours, or about 1 to 72 hours, orabout 1 to 48 hours, or about 10 to 30 hours, or about 15 to 25 hours,or about 20 hours.

In some embodiments of each of the methods and compositions describedherein, the initial concentration of the quencher (e.g., glutathione) inthe mixture comprising the red blood cell composition, quencher,pathogen-inactivating compound and any added base is greater than 2 mM,greater than about 4 mM, greater than about 6 mM, greater than about 8mM, greater than about 10 mM, or greater than about 15 mM; the loweredconcentration of the quencher in the mixture following the period ofinactivation is less than about 25 mM, less than about 20 mM, less thanabout 15 mM, less than about 10 mM, less than about 8 mM, less thanabout 6 mM, less than about 5 mM, less than about 4 mM, less than about3 mM, less than about 2 mM, less than about 1.5 mM, less than about 1mM, less than about 0.75 mM, less than about 0.5 mM, or less than about0.25 mM; and the period of time between the point of addition of thequencher at the initial concentration and the point of reducing theconcentration of the quencher to a lowered concentration is greaterthan, about equal to, or less than 5 hours, 10 hours, 15 hours, 20hours, 25 hours, 30 hours, 35 hours, 40 hours, or 50 hours.

In some embodiments, the initial concentration of the quencher is in therange of about 2 mM to 100 mM, about 2 mM to 40 mM, about 4 mM to 40 mM,about 5 mM to 40 mM, about 5 mM to 30 mM, or about 10 mM to 30 mM, orabout 20 mM; the lowered concentration of the quencher in the mixturefollowing the period of inactivation is in the range of about 1 mM to 20mM, about 2 mM to 15 mM, about 3 mM to 10 mM, about 4 mM to 8 mM, orabout 5 mM to 6 mM; and the period of time between the point of additionof the quencher at the initial concentration and the point of reducingthe concentration of the quencher to a lowered concentration is about 1to 96 hours, or about 1 to 72 hours, or about 1 to 48 hours, or about 10to 30 hours, or about 4 to 30 hours, or about 10 to 25 hours, or about15 to 25 hours, or about 20 hours.

In some embodiments, the initial concentration of the quencher (e.g.,glutathione) is in the range of about 10 mM to 30 mM; the loweredconcentration of the quencher in the mixture following the period ofinactivation is in the range of about 2 mM to 15 mM; and the period oftime between the point of addition of the quencher at the initialconcentration and the point of reducing the concentration of thequencher to a lowered concentration is about 10 to 30 hours. In someembodiments, the initial concentration of the quencher (e.g.,glutathione) is about 20 mM; and the lowered concentration of thequencher in the mixture following the period of inactivation is in therange of about 4 mM to 8 mM; and the period of time between the point ofaddition of the quencher at the initial concentration and the point ofreducing the concentration of the quencher to a lowered concentration isabout 15 to 25 hours. In some of these embodiments, the pH of themixture is in the range of about 6.5 to 7.0 (or 7.1). In other of theseembodiments, the pH of the mixture is in the range of about 6.0 to 7.5.

In any of these embodiments, the temperature of the mixture comprisingthe red blood cell composition and quencher during the period of timebetween the point of addition of the quencher at the initialconcentration and the point of reducing the concentration of thequencher to a lowered concentration in is in a temperature range ofabout 1° C. to 30° C., also about 18° C. to 25° C., or about 37° C., orabout room temperature.

In some embodiments, the present invention provides a method of treatinga red blood cell composition comprising: a) providing i) apathogen-inactivating compound (e.g., an effective amount of apathogen-inactivating compound to inactivate a pathogen, if present)comprising a frangible linker linking a mustard group and a nucleicacid-binding ligand (e.g., S-303), ii) a quencher (e.g., an effectiveamount of a quencher) comprising a thiol group, wherein the thiol iscapable of reacting with the reactive electrophilic group of thepathogen-inactivating compound (e.g., glutathione), iii) a compositioncomprising red blood cells, and iv) a suitable base (e.g., NaOH); b)mixing the pathogen-inactivating compound, quencher, and suitable basewith the composition comprising red blood cells; and c) sufficientlydecreasing the concentration of the quencher in the mixture to an amountwhich reduces the level of red blood cell dehydration resulting fromstorage of the mixture (e.g., after 10, 28, or 42 days at 4° C.),relative to the level of red blood cell dehydration resulting fromstorage of the mixture at the original concentration of quencher. Insome embodiments, the mixture comprises about 0.5 to 1.5 equivalents ofbase (or about 0.75 to 1.25 equivalents), where an equivalent means amolar amount that is equivalent to the molar amount of quencher in themixture, and/or the resulting mixture of step (b) has a pH at 37° C. ofabout 6.0 to 7.5 (or about 6.5 to 7.0, or 7.1). In some embodiments, thebase of step (a) is of sufficient amount to reduce the level ofanti-pathogen inactivating compound antibody binding to the treated redblood cell composition in the resulting mixture by at least about 5% (orat least about 10%, at least about 25%, at least about 50%, at leastabout 75%, or at least about 90%) relative to the mixture without thebase. In some embodiments, the quencher concentration is about 5 mM toabout 30 mM (or about 15 mM to about 25 mM) and/or the quencher in theresulting mixture of step (c) is at a concentration of less than about10 mM (or less than about 6 mM, or less than about 2 mM). In someembodiments, the concentration of the pathogen inactivation compound inthe resulting mixture of step (b) is about 0.1 μM to about 5 mM and/oris sufficient to inactivate at least 1 log (or 3 log) of a pathogen inthe red blood cell composition, if present. In some embodiments, thetime between step (b) and step (c) is about 1 to 48 hours (or 15 to 25hours). In some embodiments, at 20 hours following step (b), the redblood cells (RBCs) of the resulting mixture have an antibody bindingcapacity (ABC) of less than 65% compared to the ABC value of red bloodcells from the same method under the same conditions, but without theuse of base and/or have an average ABC of less than about 50,000 (orbetween about 25,000 and 70,000), and/or have less then 1% hemolysisfollowing step (c) (or following storage for 28 or 42 days at 4° C.)and/or have a Packed Cell Volume (PCV) of greater than 50% followingstep (c) (or following storage for 28 or 42 days at 4° C.) and/or have aMedian Corpuscular Fragility value greater than 140 (or 150) after 28(or 42) days at 4° C. following step (c). In some of these embodiments,decreasing the concentration of the quencher in step (c) comprisesremoval of the solution used during inactivation and addition of a finaladditive solution (e.g., any solution described in herein, such asSAG-M, AS-5, any solution of Tables 2, 3, or 4, or an additive solutioncomprising sodium chloride, adenine, glucose, phosphate, guanosine,citrate, and/or mannitol).

In some embodiments, the present invention provides a method of treatinga red blood cell composition comprising (a) mixing (i) apathogen-inactivating compound (e.g., an effective amount of apathogen-inactivating compound to inactivate a pathogen, if present)comprising a functional group which is, or which forms, a reactiveelectrophilic group (e.g., S-303); (ii) a quencher (e.g., an effectiveamount of a quencher) comprising a thiol group (e.g., glutathione),wherein the thiol is capable of reacting with the reactive electrophilicgroup of the pathogen-inactivating compound; (iii) a compositioncomprising red blood cells; and (iv) a suitable base (e.g., NaOH), and;(b) sufficiently decreasing the concentration of the quencher in themixture to an amount which reduces the level of red blood celldehydration resulting from storage of the mixture relative to the levelof red blood cell dehydration resulting from storage of the mixture(e.g., after 10, 28, or 42 days at 4° C.) at the original concentrationof quencher. In some embodiments, the mixture comprises about 0.5 to 1.5equivalents of base (or about 0.75 to 1.25 equivalents), where anequivalent means a molar amount that is equivalent to the molar amountof quencher in the mixture, and/or the resulting mixture of step (a) hasa pH at 37° (of about 6.0 to 7.5 (or about 6.5 to 7.0, or 7.1). In someembodiments, the base of step (a) is of sufficient amount to reduce thelevel of anti-pathogen inactivating compound antibody binding to thetreated red blood cell composition in the resulting mixture by at leastabout 5% (or at least about 10%, at least about 25%, at least about 50%,at least about 75%, or at least about 90%) relative to the mixturewithout the base. In some embodiments, the quencher concentration isabout 5 mM to about 30 mM (or about 15 mM to about 25 mM) and/or thequencher in the resulting mixture of step (b) is at a concentration ofless than about 10 mM (or less than about 6 mM, or less than about 2mM). In some embodiments, the concentration of the pathogen inactivationcompound in the resulting mixture of step (a) is about 0.1 M to about 5mM and/or is sufficient to inactivate at least 1 log (or 3 log) of apathogen in the red blood cell composition, if present. In someembodiments, the time between step (a) and step (b) is about 1 to 48hours (or 15 to 25 hours). In some embodiments, at 20 hours followingstep (a), the red blood cells (RBCs) of the resulting mixture have anantibody binding capacity (ABC) of less than 65% compared to the ABCvalue of red blood cells from the same method under the same conditions,but without the use of base and/or have an average ABC of less thanabout 50,000 (or between about 25,000 and 70,000), and/or have less then1% hemolysis following step (b) (or following storage for 28 or 42 daysat 4 (C) and/or have a Packed Cell Volume (PCV) of greater than 50%following step (b) (or following storage for 28 or 42 days at 4° C.)and/or have a Median Corpuscular Fragility value greater than 140 (or150) after 28 (or 42) days at 4 (C following step (b). In some of theseembodiments, decreasing the concentration of the quencher in step (b)comprises removal of the solution used during inactivation and additionof a final additive solution (e.g., any solution described in herein,such as SAG-M, AS-5, any solution of Tables 2, 3, or 4, or an additivesolution comprising sodium chloride, adenine, glucose, phosphate,guanosine, citrate, and/or mannitol).

In some embodiments, the present invention provides a method of reducingdehydration in red blood cells, comprising: a) providing a red bloodcell composition comprising i) a quencher (e.g., glutathione), where thequencher is capable of reacting with a pathogen-inactivating compound,and ii) red blood cells; and b) sufficiently decreasing theconcentration of the quencher in the mixture to an amount which reducesthe level of red blood cell dehydration resulting from storage of themixture relative to the level of red blood cell dehydration resultingfrom storage of the mixture at the original concentration of quencher(e.g., after 10, 28, or 42 days at 4° C.). In some embodiments, thequencher in the resulting mixture of step (b) is at a concentration ofless than about 10 mM (or less than about 6 mM, or less than about 2mM). In some embodiments, the red blood cells (RBCs) of the resultingmixture have less then 1 hemolysis following step (b) (or followingstorage for 28 or 42 days at 4° C.) and/or have a Packed Cell Volume(PCV) of greater than 50% following step (b) (or following storage for28 or 42 days at 4° C.) and/or have a Median Corpuscular Fragility valuegreater than 140 (or 150) after 28 (or 42) days at 4° C. following step(b).

The methods of the invention include the ex vivo use of apathogen-inactivating compound and a quencher. The ex vivo use involvesusing the compounds for treatment of a red blood cell composition,outside of a living human, mammal, or vertebrate, where the treatedbiological material is intended for use inside of a living human,mammal, or vertebrate. For example, removal of blood from a human, andintroduction of a compound into that blood to inactivate pathogens, isdefined as an ex vivo use of the compound if the blood is intended forreintroduction into that human or another human. Reintroduction of thehuman blood into that human or another human would be in vivo use of theblood, as opposed to the ex vivo use of the compound. If the compound isstill present in the blood when it is reintroduced into the human, thenthe compound, in addition to its ex vivo use, is also introduced invivo. Some embodiments of the present invention involve the ex vivo useof a quencher, where the red blood cell composition is intended for invivo use. In some instances, some level of quencher remains in the redblood cell composition such that the quencher is also introduced invivo. The in vitro use of a material or compound involves a use of thematerial or compound outside of a living human, mammal, or vertebrate,where the material or compound is not intended for reintroduction into aliving human, mammal, or vertebrate. An example of an in vitro use wouldbe the diagnostic analysis of components of a red blood cell sample. Themethods of the invention may be applied to the in vitro use of the redblood cell compositions, as modification of the red blood cells or otherconstituents may affect the in vitro analysis of the components of theblood sample. Thus, the methods of the invention may provide safety inhandling of such in vitro samples with adequate quenching ofmodifications of the sample that might otherwise interfere withdiagnostic testing of the sample.

Additive solutions, including salts and/or buffered solutions, may beused with the methods and red blood cell compositions described herein.For example, a selected buffer (e.g., SAG-M, AS-5, or any solutiondescribed in Tables 2, 3, and/or 4) may be added to the red blood cellcomposition prior to, during, and/or following the period ofinactivation and/or at the time the quencher concentration is decreased.

Methods of Inactivation Using Packed Red Blood Cells

In some embodiments, packed red blood cells (pRBCs) (e.g., red bloodcells lacking additive solution and/or having a hematocrit in the rangeof about 70 to 90%, or about 75 to 85%, or about 80%) are subjected toan inactivation method described herein (e.g., a method wherein thecomposition comprises about 20 mM GSH with about 1 equivalent base andabout 0.2 mM S-303), then subjected to (in some cases, preserved with)an additive solution (e.g., SAG-M, AS-5, or any solution describedherein or in Table 2). Examples of additive solutions are shown in Table2 and described herein. In some of these embodiments, the additivesolution (e.g., any solution described herein or in Table 2) is added tothe red blood cell composition comprising quencher,pathogen-inactivating compound, and any added base, from about 5 minutesto 20 hours following the addition of the pathogen-inactivating compound(e.g., S-303) and/or the quencher (e.g., GSH). In some embodiments, theadditive solution is added to the RBC composition from about 5 minutesto 10 hours, or about 5 minutes to 5 hours, or about 5 minutes to 60minutes, or about 5 minutes to 30 minutes, or about 10 minutes to 20minutes, or about 15 minutes following the addition of thepathogen-inactivating compound (e.g., S-303) and/or the quencher (e.g.,GSH). In some embodiments, the concentration of the quencher isdecreased as described herein following the addition of the additivesolution (e.g., SAG-M, AS-5, or any solution described herein or inTable 2). For example, pRBCs may be treated with an inactivation methoddescribed herein (e.g., treatment wherein the composition comprisesabout 20 mM GSH, about 1 equivalent base, and about 0.2 mM S-303), thentreated with an additive solution (e.g., SAG-M, AS-5, or any solutiondescribed herein or in Table 2) at a specified time after the additionof the pathogen-inactivating compound and/or the quencher (such as atabout 5 minutes to 5 hours, or about 10 minutes to 20 minutes, or about15 minutes), followed by decreasing of the quencher concentration asdescribed herein (e.g., to less than about 10 mM, or less than about 5mM). In some of these embodiments, decreasing the quencher concentrationcomprises removal of the treatment solution and/or additive solution,followed by the addition of a final additive solution (e.g., SAG-M,AS-5, or any solution described herein or in Table 2) to provide a redblood cell composition having, e.g., a hematocrit in the range about 50to 70%, or about 55 to 65%, or about 60%. In some embodiments, theconcentration of chloride ion in the red blood cell composition prior toand/or during inactivation is less than or greater than about 150 mM, orabout 120 mM, or about 100 mM, or about 90 mM, about 80 mM, or about 70mM, or about 60 mM, or about 50 mM, about 40 mM, about 30 mM, or about20 mM, about 10 mM, or between about 25 and 250 mM, or about 40 and 100mM, or about 50 and 75 mM, or about 60 and 70 mM, or about 65 mM.

In some embodiments, the additive solution referred to herein (e.g., theadditive solution administered prior to and/or following decrease of thequencher concentration) comprises one or more of the followingcomponents: dextrose, adenine, guanosine, mannitol, citrate (e.g.,sodium citrate), citric acid, phosphate (e.g., Na₂HPO₄ and/or NaH₂PO₄)and chloride (e.g., from sodium chloride). In some embodiments, theconcentration of dextrose of the additive solution and/or the finalconcentration of dextrose in the RBC composition following exchange(e.g., prior to transfusion) is at a concentration from about 10 mM toabout 150 mM, or about 20 mM to about 120 mM, or about 25 mM to about100 mM, or about 30 mM to about 75 mM, or about 40 mM to about 50 mM. Insome embodiments, the concentration of adenine of the additive solutionand/or the final concentration of adenine in the RBC compositionfollowing exchange (e.g., prior to transfusion) is at a concentrationfrom about 0.5 mM to about 5 mM, or about 0.75 mM to about 3 mM, orabout 1 mM to about 2.5 mM. In some embodiments, the concentration ofguanosine of the additive solution and/or the final concentration ofguanosine in the RBC composition following exchange (e.g., prior totransfusion) is at a concentration from about 0.5 mM to about 5 mM, orabout 0.75 mM to about 3 mM, or about 1 mM to about 2.5 mM, or about 1.5mM to about 2 mM. In some embodiments, the concentration of mannitol ofthe additive solution and/or the final concentration of mannitol in theRBC composition following exchange (e.g., prior to transfusion) is at aconcentration from about 10 mM to about 150 mM, or about 20 mM to about120 mM, or about 25 mM to about 100 mM, or about 30 mM to about 75 mM,or about 40 mM to about 50 mM, or about 35 mM to about 45 mM. In someembodiments, the concentration of citrate (e.g., sodium citrate) of theadditive solution and/or the final concentration of citrate in the RBCcomposition following exchange (e.g., prior to transfusion) is at aconcentration from about 5 mM to about 100 mM, or about 10 mM to about75 mM, or about 15 mM to about 50 mM, or about 15 mM to about 35 mM, orabout 20 mM to about 30 mM. In some embodiments, the concentration ofphosphate (e.g., Na₂HPO₄ and/or NaH₂PO₄) of the additive solution and/orthe final concentration of phosphate in the RBC composition followingexchange (e.g., prior to transfusion) is at a concentration from about 1mM to about 150 mM, or about 2 mM to about 100 mM, or about 3 mM toabout 75 mM, or about 4 mM to about 50 mM, or about 5 mM to about 25 mM,or about 10 mM to about 20 mM. In some embodiments, the concentration ofchloride of the additive solution and/or the final concentration ofchloride in the RBC composition following exchange (e.g., prior totransfusion) is less than or greater than about 500 mM, or about 250 mM,or about 200 mM, or about 150 mM, or about 100 mM, about 75 mM, or about50 mM, or about 25 mM, or about 25 to about 250 mM, or about 40 to about100 mM, or about 50 to about 75 mM, or about 60 to about 70 mM, or about100 to about 200 mM, or about 125 mM to about 175 mM, or about 150 mM.

In some embodiments, the additive solution referred to herein (e.g., theadditive solution administered prior to and/or following decrease of thequencher concentration) and/or the final RBC composition followingexchange (e.g., prior to transfusion) comprises 10 mM to about 150 mM(or about 50 mM to about 90 mM) dextrose, 0.5 mM to about 5 mM (or about0.75 mM to about 3 mM) adenine, about 10 mM to about 150 mM (or about 25mM to about 100 mM) mannitol, about 10 mM to about 75 mM (or about 15 mMto about 50 mM) citrate (e.g., sodium citrate), about 3 mM to about 75mM (or about 5 mM to about 25 mM) phosphate (e.g., Na₂HPO₄ and/orNaH₂PO₄), and about 50 to about 250 mM, or (about 100 to about 175 mM)chloride.

TABLE 2 Exemplary Additive Solutions SAG- Eryth- PAGGS- AS-1 AS-3 Mrosol AS-5 M MAP Dextrose 111.0 55.5 45.4 81.1 45.4 47.5 36.4 (mM)Adenine 2.0 2.2 1.3 1.6 2.2 1.4 1 (mM) Guanosine 1.44 (mM) Mannitol 41.228.8 42.5 28.8 55 80 (mM) Sodium 20 26.6 5.1 Citrate Dihydrate (mM)Na₂HPO₄ 17 8 (mM) NaH₂PO₄ 20 4.7 8 6 (mM) NaCl (mM) 154.0 70 150 150 7285 Citric Acid 2 1 (mM) Osmolality 276 359 175 351 296 (mOsm)

Methods of Inactivation Using Diluted Red Blood Cells

The red blood cell compositions described herein may be diluted prior toinactivation. Subjecting the red blood cells to a diluent may decreasethe concentration of dissolved species (e.g., salts such as Cl⁻) to alevel that is suitable for the inactivation with methods describedherein. Examples of diluent solutions are described herein and shown inTable 3. In some embodiments, non-packed red blood cells (e.g., redblood cells having a hematocrit in the range about 50 to 70%, or about55 to 65%, or about 60% and optionally comprising SAG-M or Optisol) aresubjected to a diluent solution (e.g., any solution described herein orin Table 3) prior to an inactivation method described herein (e.g., amethod wherein the composition comprises about 20 mM GSH with about 1equivalent base and about 0.2 mM S-303), followed by decreasing of thequencher concentration as described herein (e.g., to less than about 10mM, or less than about 5 mM). In some of these embodiments, decreasingthe quencher concentration comprises removal of the treatment solution(e.g., a diluted treatment solution) followed by the addition of a finaladditive solution (e.g., SAG-M, AS-5, or any solution described above orin Table 2) to provide, for example, a red blood cell composition havinga hematocrit in the range about 50 to 70%, or about 55 to 65%, or about60%). In some embodiments, the concentration of chloride ion in the redblood cell composition is diluted to less than or greater than about 150mM, or about 120 mM, or about 100 mM, or about 90 mM, about 80 mM, orabout 70 mM, or about 60 mM, or about 50 mM, about 40 mM, about 30 mM,or about 20 mM, about 10 mM, or between about 25 and 250 mM, or about 40and 100 mM, or about 50 and 75 mM, or about 60 and 70 mM, or about 65 mMprior to inactivation. In some embodiments, the amount (by volume) ofdiluent solution added to the RBC solution is between about 0.2 and 2times, or about 0.3 and 1.5 times, or about 0.4 and 1 times, or about0.5 and 0.75 times the amount of RBC solution. In some of theseembodiments, the red blood cell composition is diluted with a diluentsolution (e.g., any solution described herein or in Table 3) to ahematocrit level in the range about 30 to 50%, or about 35 to 45%, orabout 40%.

In some embodiments, the diluent solution referred to herein comprisesone or more of the following components: dextrose, adenine, mannitol,citrate (e.g., sodium citrate), citric acid, phosphate (e.g., Na₂HPO₄and/or NaH₂PO₄) and chloride (e.g., from sodium chloride). In someembodiments, the concentration of dextrose of the diluent solutionand/or the final concentration of dextrose in the RBC compositionfollowing dilution with the diluent solution is at a concentration fromabout 10 mM to about 150 mM, or about 20 mM to about 120 mM, or about 25mM to about 100 mM, or about 30 mM to about 75 mM, or about 40 mM toabout 50 mM, or about 50 mM to about 60 mM. In some embodiments, theconcentration of adenine of the diluent solution and/or the finalconcentration of adenine in the RBC composition following dilution withthe diluent solution is from about 0.5 mM to about 5 mM, or about 0.75mM to about 3 mM, or about 1 mM to about 2.5 mM. In some embodiments,the concentration of mannitol of the diluent solution and/or the finalconcentration of mannitol in the RBC composition following dilution isfrom about 10 mM to about 150 mM, or about 20 mM to about 120 mM, orabout 25 mM to about 100 mM, or about 30 mM to about 75 mM, or about 40mM to about 60 mM, or about 25 mM to about 35 mM. In some embodiments,the concentration of citrate (e.g., sodium citrate) of the diluentsolution and/or the final concentration of citrate in the RBCcomposition following dilution with the diluent solution is from about 5mM to about 100 mM, or about 10 mM to about 75 mM, or about 15 mM toabout 50 mM, or about 15 mM to about 35 mM, or about 20 mM to about 30mM. In some embodiments, the concentration of phosphate (e.g., Na₂HPO₄and/or NaH₂PO₄) of the diluent solution and/or the final concentrationof phosphate in the RBC composition following dilution with the diluentsolution is from about 1 mM to about 150 mM, or about 2 mM to about 100mM, or about 3 mM to about 75 mM, or about 4 mM to about 50 mM, or about5 mM to about 25 mM, or about 10 mM to about 20 mM. In some embodiments,the concentration of chloride of the diluent solution and/or followingdilution with the diluent solution is less than or greater than about500 mM, or about 250 mM, or about 200 mM, or about 150 mM, or about 120mM, or about 100 mM, or about 90 mM, or about 80 mM, or about 70 mM, orabout 60 mM, or about 50 mM, or about 40 mM, or about 30 mM, or about 20mM, about 10 mM or about 25 to about 250 mM, or about 40 to about 100mM, or about 50 to about 75 mM, or about 60 to about 70 mM, or about 100to about 200 mM, or about 125 mM to about 175 mM.

In some embodiments, the diluent solution referred to herein and/or theRBC composition following dilution with the diluent solution comprises10 mM to about 150 mM (or about 35 mM to about 65 mM) dextrose, 0.5 mMto about 5 mM (or about 0.75 mM to about 3 mM) adenine, about 10 mM toabout 150 mM (or about 25 mM to about 75 mM) mannitol, about 10 mM toabout 75 mM (or about 15 mM to about 50 mM) citrate (e.g., sodiumcitrate), about 3 mM to about 75 mM (or about 5 mM to about 25 mM)phosphate (e.g., Na₂HPO₄ and/or NaH₂PO₄), and about 5 to about 50 mM, or(about 10 to about 25 mM) chloride.

In some embodiments, non-packed red blood cells are subjected to adiluent solution (e.g., any solution described above or in Table 3)prior to an inactivation method described herein, followed by decreasingof the quencher concentration as described herein, then treated with afinal additive solution (e.g., SAG-M, AS-5, or any solution describedabove or in Table 2) to provide an RBC composition suitable for use(e.g., suitable of transfusion). In some embodiments, the final additivesolution may be any additive solution described herein, for example, anadditive solution wherein the concentration of chloride (and/or thefinal concentration of chloride in the RBC composition followingexchange, such as prior to transfusion) is less than about 500 mM, orabout 250 mM, or about 200 mM, or about 150 mM, or about 100 mM, about75 mM, or about 50 mM, or about 25 mM, or between about 25 and 250 mM,or about 40 and 100 mM, or about 50 and 75 mM, or about 60 and 70 mM, orabout 100 and 200 mM, or about 125 mM and 175 mM, or about 150 mM.

TABLE 3 Exemplary Diluent Solutions DS DS DS DS DS DS DS DS DS DS DS DSDS 1 2 3 4 5 6 7 8 9 10 11 12 13 Dextrose 55 55 55 55 55 45.4 45.4 45.445.4 45.4 45.4 45.4 (mM) Adenine/ 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.31.3 1.3 1.3 1.3 Adenine HCl (mM) Mannitol 55 55 55 55 55 28.8 28.8 28.828.8 28.8 28.8 (mM) Sodium 20 20 33.5 33.5 33.5 20 20 20 20 20 Citratedihydrate or anhydrous (mM) Na₂HPO₄ 20 15 33.5 12.7 3.5 16.2 20 (mM)NaH₂PO₄ 33.5 33.5 3.5 12.7 16.2 (mM) NaCl (mM) 15 20 Osmolality 180 178128 177 179 287 243 215 227 179 174 181 (mOsm) pH 6.9 7.6-8.0 8.61 8.386.28 6.29 8.77 7.48 6.63 8.62 8.7 7.5

Methods of Inactivation Using Reconstituted Packed Red Blood Cells

In some embodiments, packed red blood cells (pRBCs) (e.g., red bloodcells having a hematocrit in the range of about 70 to 90%, or about 75to 85%, or about 80%) are subjected to a treatment solution prior toconducting the inactivation method described herein (e.g., a methodwherein the composition comprises about 20 mM GSH with about 1equivalent base and about 0.2 mM S-303). Examples of treatment solutionsare shown in Table 4. In some embodiments, the treatment solution (e.g.,any solution described in Table 4) is added to the pRBCs prior to theaddition of the quencher, pathogen-inactivating compound, and any addedbase. In some of these embodiments, the pRBC composition is treated witha treatment solution resulting in non-packed red blood cells (e.g., redblood cells having a hematocrit in the range about 50 to 70%, or about55 to 65%, or about 60%). In some embodiments, (a) a treatment solutionis added to pRBCs, (b) an inactivation method described herein (e.g., amethod wherein the composition comprises about 20 mM GSH with about 1equivalent base and about 0.2 mM S-303) is conducted, and (c) theconcentration of the quencher is decreased as described herein (e.g., toless than about 10 mM, or less than about 5 mM). In some of theseembodiments, step (c) comprises removal of the treatment solution andaddition of a final additive solution (e.g., any solution described inherein, such as SAC-M, AS-5 or any solution of Tables 2, 3, or 4) toprovide, for example, a red blood cell composition having a hematocritin the range about 50 to 70%, or about 55 to 65%, or about 60%. In someof these embodiments, the concentration of chloride ion in the red bloodcell composition prior to and/or during inactivation is less than orgreater than about 150 mM, or about 120 mM, or about 100 mM, or about 90mM, about 80 mM, or about 70 mM, or about 60 mM, or about 50 mM, about40 mM, about 30 mM, or about 20 mM, about 10 mM, or between about 25 and250 mM, or about 40 and 100 mM, or about 50 and 75 mM, or about 60 and70 mM, or about 65 mM.

In some embodiments, the treatment solution referred to herein comprisesone or more of the following components: dextrose, adenine, mannitol,citrate (e.g., sodium citrate), citric acid, phosphate (e.g., Na₂HPO₄and/or NaH₂PO₄) and chloride (e.g., from sodium chloride). In someembodiments, the concentration of dextrose of the treatment solutionand/or the concentration of dextrose in the additive solution followingremoval of the treatment solution in the RBC composition is from about10 mM to about 150 mM, or about 20 mM to about 120 mM, or about 25 mM toabout 100 mM, or about 30 mM to about 75 mM, or about 40 mM to about 50mM, or about 50 mM to about 60 mM. In some embodiments, theconcentration of adenine of the treatment solution and/or theconcentration of adenine in the additive solution following removal ofthe treatment solution in the RBC composition is from about 0.5 mM toabout 5 mM, or about 0.75 mM to about 3 mM, or about 1 mM to about 2.5mM. In some embodiments, the concentration of mannitol in the treatmentsolution and/or the concentration of mannitol in the additive solutionfollowing removal of the treatment solution in the RBC composition isfrom about 10 mM to about 150 mM, or about 20 mM to about 120 mM, orabout 25 mM to about 100 mM, or about 30 mM to about 75 mM, or about 40mM to about 60 mM. In some embodiments, the concentration of citrate(e.g., sodium citrate) in the treatment solution and/or theconcentration of citrate in the additive solution following removal ofthe treatment solution in the RBC composition is from about 1 mM toabout 100 mM, or about 2 mM to about 75 mM, or about 5 mM to about 50mM, or about 7.5 mM to about 25 mM, or about 10 mM to about 15 mM. Insome embodiments, the concentration of phosphate (e.g., Na₂HPO₄ and/orNaH₂PO₄) in the treatment solution and/or the concentration of phosphatein the additive solution following removal of the treatment solution inthe RBC composition is from about 1 mM to about 150 mM, or about 2 mM toabout 100 mM, or about 3 mM to about 75 mM, or about 4 mM to about 50mM, or about 5 mM to about 25 mM, or about 10 mM to about 20 mM. In someembodiments, the concentration of chloride in the treatment solutionand/or the concentration of chloride in the additive solution followingremoval of the treatment solution in the RBC composition is from about250 mM, or about 200 mM, or about 150 mM, or about 120 mM, or about 100mM, or about 90 mM, or about 80 mM, or about 70 mM, or about 60 mM, orabout 50 mM, or about 40 mM, or about 30 mM, or about 20 mM, about 10mM, or about 25 to about 250 mM, or about 40 to about 100 mM, or about50 to about 75 mM, or about 60 to about 70 mM, or about 100 to about 200mM, or about 125 mM to about 175 mM.

In some embodiments, the treatment solution and/or the additive solutionfollowing removal of the treatment solution in the RBC compositioncomprises 10 mM to about 150 mM (or about 35 mM to about 65 mM)dextrose, 0.5 mM to about 5 mM (or about 0.75 mM to about 3 mM) adenine,about 10 mM to about 150 mM (or about 25 mM to about 75 mM) mannitol,about 5 mM to about 75 mM (or about 10 mM to about 20 mM) citrate (e.g.,sodium citrate), about 3 mM to about 75 mM (or about 5 mM to about 25mM) phosphate (e.g., Na₂HPO₄ and/or NaH₂PO₄), and about 5 to about 100mM, or (about 25 to about 75 mM) chloride.

TABLE 4 Exemplary Treatment Solutions Sol 1 Sol 2 Sol 3 Sol 4 Sol 5Dextrose (mM) 45.4 45.4 45.4 45.4 45.4 Adenine/Adenine 1.3 1.3 1.3 1.31.3 HCl(mM) Mannitol (mM) 55 44.5 44.5 44.5 30 Sodium Citrate 12 12 12dihydrate or anhydrous (mM) Na₂HPO₄ (mM) 15 NaH₂PO₄ (mM) NaCl (mM) 70 6060 60 70 Osmolality (mOsm) pH (adjusted with 7 6.5 6.5 citric acid)Evaluating Method Efficacy

In addition to comparing the log inactivation as discussed above, theefficacy of the improved quenching methods may be evaluated by severalother methods, as described in US Patent Publication No. 2006/0115466,the content of which is hereby incorporated by reference in itsentirety. For example, the quenching methods may be assessed byevaluating the modification of the red blood cell composition, in termsof the function, morphology and the hydration status of the red bloodcells, and in terms of the reactivity of the treated red blood cellswith the immune system, such as with antibodies. If the treated redblood cell composition is intended for human use, such as infusion, thequenching methods should not substantially damage red blood cellfunction (e.g., via dehydration). The lack of a substantially damagingeffect on red blood cell function may be measured by methods known inthe art for testing red blood cell function. In particular, levels ofdehydration can be measured, for example, by hematocrit (packed cellvolume, PCV), osmotic fragility, mean corpuscular hemoglobinconcentration (MCHC), percent hemolysis, and ektacytometry. The levelsof other indicators of function, such as total ATP (adenosine5′-triphosphate), total 2,3-DPG (2,3-diphosphoglycerol) or extracellularpotassium may be measured, and compared to an untreated control.Additionally, intracellular and extracellular pH, hemoglobin, glucoseconsumption and lactate production may be measured. The improved methodsof the present invention can be compared to the previously describedconditions of treatment in US Patent Publication No. 2006/0115466 (e.g.,fully quenched (2 base equivalent) 20 mM glutathione in combination withthe S-303/red blood cell mixture without reduction in quencherconcentration following incubation described therein).

In some embodiments of the present invention, the red blood cells of themethods and compositions described herein have minimal or no damagefollowing treatment (e.g., dehydration, hemolysis, etc.). In someembodiments, the red blood cells of the resulting mixture comprising thered blood cell composition, quencher, pathogen-inactivating compound andany added base (before or after the reduction in the quencherconcentration) have less than 4%, or less than 3%, or less than 2%, lessthan 1% hemolysis, or less than 0.5% hemolysis. In some embodiments, thered blood cells of the resulting mixture have less than 4%, or less than3%, or less than 2%, or less than 1%, or less than 0.5% hemolysis at atime of about 10 days at 4° C., or about 28 or 42 days at 4° C., orabout 42 days at 4° C. following the reduction in concentration of thequencher (e.g., glutathione).

In some embodiments, the red blood cells of the resulting mixturecomprising the red blood cell composition, quencher,pathogen-inactivating compound and any added base (before or after thereduction in the quencher concentration) have greater than 50%, orgreater than 55%, or greater than 60%, or greater than 65% packed cellvolume (PCV). In some embodiments, the red blood cells of the resultingmixture have greater than 50%, or greater than 55%, or greater than 60%,or greater than 65% packed cell volume (PCV) at a time of about 10 daysat 4° C., or about 28 or 42 days at 4° C., or about 42 days at 4° C.following the reduction in concentration of the quencher (e.g.,glutathione).

In some embodiments, the red blood cells of the resulting mixturecomprising the red blood cell composition, quencher,pathogen-inactivating compound and any added base (before or after thereduction in the quencher concentration) have a Median CorpuscularFragility (MCF; osmolarity at which 50% of hemolysis occurs) greaterthan 130, or greater than 135, or greater than 140, or greater than 145,or greater than 150, or greater than 155. In some embodiments, the redblood cells of the resulting mixture have a Median Corpuscular Fragility(MCF) greater than 130, or greater than 135, greater than 140, orgreater than 145, or greater than 150, or greater than 155 at a time ofabout 10 days at 4° C., or about 28 or 42 days at 4° C., or about 42days at 4° C. following the reduction in concentration of the quencher(e.g., glutathione).

Methods for determining ATP, 2,3-DPG, glucose, hemoglobin, hemolysis,and potassium are available in the art and described herein in theexperimental section. See for example, Davey et al., Transfusion,32:525-528 (1992), the disclosure of which is incorporated herein.Methods for determining red blood cell function are also described inGreenwalt et al., Vox Sang, 58:94-99 (1990); Hogman et al., Vox Sang,65:271-278 (1993) and Beutler et al., Blood, Vol. 59 (1982) thedisclosures of which are incorporated herein by reference. For example,total ATP and total 2,3-DPG may be measured using a Sigma ATP kit or2,3-DPG; kit (Sigma, St. Louis, Mo.). The ATP kit may be used followingSigma procedure No. 366-UV, the disclosure of which is herebyincorporated by reference. Total ATP may also be measured using aluciferase based enzymatic assay or a protocol described by Beutler(1984). Extracellular potassium levels may be measured using a CibaCorning Model 614 K⁺/Na⁺ Analyzer (Ciba Corning Diagnostics Corp.,Medford, Mass.). The extracellular pH may be measured by centrifugingthe cells at 4° C. for 15 minutes at 12,000×g and removing thesupernatant, for which the pH may be measured using a standard pH meterat room temperature (e.g. Beckman, Epoxy Calomel electrode). For theintracellular pH, the remaining pellet may be capped in the centrifugetube and stored at about −80° C. for at least 2 hours. This then may belysed by the addition of deionized water. The lysed sample may be mixedwell and the pH of the solution may be measured either at roomtemperature using a standard pH meter or at room temperature using aCiba Corning Model 238 Blood Gas Analyzer (Ciba Corning DiagnosticsCorp., Medford, Mass.). Measurements can be made shortly after treatmentand as a function of post-treatment storage, for example storage for upto 42 days. The methods of the present invention provide a red bloodcell composition wherein hemolysis of the treated red blood cells isless than 3% after 28 day storage, more preferably less than 2% after 42day storage, and most preferably less than or equal to about 1% after 42day storage at 4° C. In some embodiments are provided a red blood cellcomposition (e.g., a red blood cell composition using any of the methodsdescribed herein) wherein the total ATP level may be higher whencompared to a red blood cell composition treated using 2 mM acidicglutathione and 0.2 mM S-303. In some embodiments, the quenching methodsdescribed herein provide red blood cell compositions having ATP levelsthat are about 20%, also 30%, also 40% or about 50% higher when comparedto compositions from methods using 2 mM acidic glutathione and 0.2 mMS-303. In some embodiments, the higher level of ATP is maintained after7, 14, 21, 28, 35, or 42 days of storage. In some embodiments, thehigher level of ATP decreases during storage.

In some embodiments of the present invention, the methods andcompositions described herein include red blood cell compositionswherein the red blood cells have a reduced number of unwanted sidereactions from the pathogen-inactivating compound (e.g., binding of thepathogen-inactivating compound to the RBC surface). In some embodiments,the side reaction is modification of the surface of the red blood cellsby the pathogen-inactivating compound. The reduction in modification ofred blood cells in the methods of the present invention can be evaluatedby several assays known in the art, such as those described in U.S.Patent Publication No. 2006/0115466, the content of which is herebyincorporated by reference. Quantification of acridine bound to the RBCsurface can also be determined using a sensitive fluorescence-activatedimmune flow cytometric assay (IFC) described herein.

With respect to the fluorescence detection assays, the quenching methodsof the present invention, when compared with the same treatment withoutthe use of base (e.g., methods using neutralizing glutathione comparedto the same methods using non-neutralized glutathione), may result inreduction of the median fluorescence by at least 10%, also at least 25%,also at least 50%, also at least 75%, or at least 90%. For example, thequenching methods of the present invention using any of the compositionsdescribed with the use of base (e.g., a red blood cell compositioncomprising about 15-25 mM glutathione, about 0.5 to 1.5 equivalent ofbase, and about 0.2 mM S-303) may result in a lower level of medianfluorescence when compared to an identical composition, but without theuse of base (e.g., a red blood cell composition comprising about 15-25mM glutathione and about 0.2 mM S-303 without base).

The level of pathogen-inactivating compound bound to the RBC surface forthe quenching methods and compositions of the present inventions alsocan be measured in terms of Antibody Binding Capacity (ABC; the numberof molecules of pathogen-inactivating compound or derivative thereof perred cell, as determined by the use of calibration beads from BangsLaboratories, Inc; Fishers, Ind., see Examples 5 and 9) which involves amouse monoclonal anti-acridine antibody conjugated to allophycocyanin(APC) and a FACS-Caliber flow cytometer (BD Biosciences). In someembodiments of any of the methods and compositions of the presentinvention, the RBCs have an average ABC value of less than about 75,000,or less than about 70,000, or less than about 60,000, or less than about55,000, or less than about 52,500, or less than about 50,000, or lessthan about 47,500, or less than about 45,000, or less than about 42,500,or less than about 40,000, or less than about 37,500, or less than about35,000, or less than about 32,500, or less than about 30,000, or lessthan about 27,500, or less than about 25,000. In some embodiments, theRBCs have an average ABC value of between about 10,000 and 80,000, orbetween about 20,000 and 70,000, or between about 25,000 and 70,000, orbetween about 25,000 and 60,000, or between about 30,000 and 50,000, orbetween about 35,000 and 45,000. In some embodiment of the methodsdescribed herein, when compared with such similar treatment withquencher and base (e.g., neutralized glutathione), may result in an ABCvalue of less than 90%, also less than 75%, also less than 65%, alsoless than 55%, also less than 45%, also less than 35%, also less than25%, or less than 10% as compared to the identical methods using a RBCcomposition that is not treated with base (e.g., glutathione that is notneutralized).

The quenching methods of the invention can also be compared to existingmethods by determining the level of modification of nucleic acids in asample. Typically, a red blood cell composition may contain leukocytes,and the nucleic acid from the leukocytes can be isolated. Apathogen-inactivating compound having a radioactive isotope that, uponreaction of the compound with nucleic acid, will remain bound to thenucleic acid. This can be used to assess the amount of compound reactedwith the nucleic acid for a variety of quenching methods, and provides ameasure that can be directly correlated to expected leukocyteinactivation. The number of S-303 adducts formed per 1,000 nucleic acidbase pairs can be used as a model to assess the expected impact of thevarious methods on pathogen inactivation. Alternatively, a suitableamount of a pathogen can be added to a red blood cell composition andthe nucleic acid of the pathogen can be isolated after treatment.However, in this case the sample needs to be leukoreduced such that thelevels of any residual leukocytes will not interfere with themeasurement of pathogen nucleic acid.

In addition to providing adequate pathogen inactivation while reducingthe levels of unwanted side reactions (e.g., binding of thepathogen-inactivating compound to the RBC surface which may lead to anundesired immune response) and dehydration, the quenching methods of thepresent invention also provide, in at least some embodiments, areduction in the concentration of reactive electrophilic species afterpathogen inactivation. If the red blood cell compositions are intendedfor infusion, it is important that the level of reactive electrophilicspecies is as low as possible, preferably essentially no longerdetectable. The presence of the reactive electrophilic species may bedetermined using methods available in the art, such as chromatographicmethods including liquid chromatography-mass spectroscopy (LC-MS-MS). Inaddition, the residual activity of a sample may be assessed byevaluating its ability to react with a guanine residue of a nucleicacid, such as using the general alkylator assay described by Mattes(Mattes, W R, Anal. Biochem. 1992 October; 206(1):161-7). In this assay,the RBCs are extracted after a suitable incubation time with thepathogen-inactivating compound and quencher. Any residualpathogen-inactivating compound, as well as the quencher and other smallspecies, are separated from the proteins. These species are thenincubated with double-stranded (ds) DNA synthesized with 8-³H guanineresidues. The residual pathogen-inactivating compound reacts with ds DNAat the N7 position of guanine, which acidifies the 8-H reside andreleases the ³H into solution, where it can be isolated and measured.The amount of tritium released can be quantified, and has a 1:1correlation with the amount of residual alkylator present in theextracted samples tested. The level of electrophilic species asdetermined by these methods can be assessed using the improved methodsof the invention and comparing to known methods.

In some embodiments of each of the methods described herein, the methodfurther comprises the step of reducing the concentration of a compoundin the mixture, wherein the compound is selected from the groupconsisting of the pathogen-inactivating compound and a degradationproduct of the pathogen-inactivating compound. In some embodiments, themethod comprises the step of reducing the concentration of thepathogen-inactivating compound in the mixture. In some embodiments, themethod comprises the step of reducing the concentration of theelectrophilic species in the mixture. The concentration of thepathogen-inactivating compound in a biological material, such as a bloodproduct, can be reduced after the treatment, for example by adsorptionin a batch or flow removal process. Methods and devices which may beused are described in U.S. Pat. Nos. 6,544,727; 6,331,387; 6,951,713;and 7,037,642; and U.S. Patent Applications 2002/0192632 (abandoned) and2001/0009756 (abandoned), the disclosures of each of which areincorporated herein by reference in their entirety. Accordingly, in someembodiments, the concentration of the pathogen-inactivating compound isreduced by contacting the mixture with an adsorption medium comprisingadsorbent particles having an affinity for the pathogen-inactivatingcompound. In some embodiments, the adsorption system would be configuredto remove the pathogen-inactivating compound in a batch process. In someembodiments, the pathogen inactivation compound is not reduced by usinga compound adsorption device. In some embodiments, the concentration ofthe pathogen-inactivating compound in the mixture is reduced by washingthe red blood cells using techniques known in the art. In someembodiments, the concentration of the pathogen-inactivating compound inthe mixture is reduced by removing some or all of the treatment solution(e.g., SAG-M, AS-5, or any solution described in Tables 2, 3, and/or 4)by methods described herein and/or known in the art (e.g., usingcentrifuges and expression devices or combined centrifuge and expressorssuch as TACSI made by Terumo®). In some embodiments, the concentrationof the pathogen-inactivating compound in the mixture is reduced byremoving some or all of the treatment solution (e.g., SAG-M, AS-5, orany solution described in Tables 2, 3, and/or 4), followed by additionof an additive solution (e.g., SAG-M, AS-5, or any solution described inTable 2) to the mixture. In some embodiments, the concentration of thepathogen-inactivating compound is reduced simultaneously with areduction in the concentration of the quencher.

Treated Blood Compositions

In some embodiments, the invention also provides red blood cellcompositions resulting from each of the treatment methods describedherein. In some embodiments, the invention also provides red blood cellcompositions preparable by each of the treatment methods describedherein. In one aspect, the invention provides a composition comprisinga) red blood cells, wherein the red blood cells have covalently reactedwith an electrophilic group of a pathogen-inactivating compound; and b)a quencher comprising a thiol group that is capable of reacting with thepathogen-inactivating compound; wherein the composition is suitable forinfusion into humans after storage of 28 or 42 days at 4° C.

In some embodiments of each of the methods and compositions describedherein, the red blood cells in the red blood cell composition aremammalian blood cells. For instance, the red blood cells may be rodent(e.g., mouse or rat), canine, lagomorph (e.g., rabbit), non-humanprimate (e.g., chimpanzee), or human red blood cells. For example, insome embodiments, the red blood cells are human. In some embodiments,the red blood cells have been leukoreduced. In some other embodiments,the red blood cells have not been leukoreduced. In some embodiments,there is a possibility that the composition comprising red blood cellsis contaminated with a pathogen. In some embodiments, the red blood cellcomposition is contaminated with a pathogen. In some embodiments, atleast 1 log, or at least 2 logs, or at least 3 logs, or at least 4 logsof pathogen in the composition is inactivated, if present.

In some embodiments, the invention embraces red blood cell compositionswherein the red blood cells have been modified with apathogen-inactivating compound (e.g., S-303), as described herein. Insome embodiments, the red blood cell compositions produced by thetreatment of the methods comprise degradation products of the pathogeninactivating compound (e.g., the reaction product of the quencher withthe pathogen inactivating compound). In some embodiments, themodification is reaction of the electrophilic group of apathogen-inactivating compound with the red blood cell surface. In someembodiments, the pathogen-inactivating compound is covalently bound tothe red blood cell surface. In some embodiments, thepathogen-inactivating compound is covalently bound to one or moreproteins on the red blood cell surface. In some embodiments, themodification is a nucleophilic group of the red blood cell reacted withthe electrophilic group of the pathogen-inactivating compound, whereinthe electrophilic group is a mustard group and the nucleophilic grouphas replaced one or more of the chlorine atoms of the mustard group. Insome embodiments, the pathogen-inactivating compound is non-covalentlybound to the red blood cell surface. In some embodiments the RBCcompositions have an average ABC value of less than 75,000, or less than70,000, or less than 60,000, or less than 55,000, or less than 52,500,or less than 50,000, or less than 47,500, or less than 45,000, or lessthan 42,500, or less than 40,000, or less than 37,500, or less than35,000, or less than 32,500, or less than 30,000, or less than 27,500,or less than 25,000. In some embodiments, the RBCs have an average ABCvalue of between about 10,000 and 80,000, or between about 20,000 and70,000, or between about 25,000 and 70,000, or between about 25,000 and60,000, or between about 30,000 and 50,000, or between about 35,000 and45,000.

In some embodiments, the red blood cell compositions comprise reducedlevels of modification of the surface of the red blood cells by thepathogen inactivating compound, relative to red blood cells produced byother methods involving treatment with the pathogen inactivatingcompound. In some embodiments, the red blood cell compositions producedby the treatments of the methods described herein comprise a reducedamount of pathogen inactivating compound comprising the reactiveelectrophilic group after completion of the treatment, relative to a redblood cells composition produced by another method involving treatmentwith the pathogen inactivating compound (e.g., a method withoutsufficient quencher and/or base added to the reaction mixture, a methodin which no quencher and/or base is added to the reaction mixture,and/or a treatment at a lower pH). In some embodiments, the amount ofpathogen inactivating compound comprising the reactive electrophilicgroup in the composition has been reduced by about 10%, about 25%, about50%, about 75%, about 90%, about 95%, or about 99%, relative to acomposition treated by another method involving the pathogeninactivating compound (e.g., a method without sufficient quencher and/orbase added to the reaction mixture, a method in which no quencher and/orbase is added to the reaction mixture, and/or treatment at a lower pH).

In some of these embodiments, the red blood cell composition comprisesresidual quencher compound (e.g., glutathione). In some embodiments, thecomposition comprises a concentration of quencher sufficiently low tomaintain RBC vitality and lifespan and avoid red blood cell dehydrationand/or reduced osmotic fragility during storage. In some embodiments,the composition comprises a concentration of quencher that issufficiently lower than a concentration of quencher previously used inthe composition. In some embodiments, the higher concentration ofquencher previously used decreases RBC vitality and lifespan and/orincreases red blood cell dehydration and decreases osmotic fragilityduring storage, while the lower concentration is sufficiently lower thana concentration of quencher previously used in the composition. In someembodiments, the concentration of the quencher in the composition isless than about 25 mM, less than about 20 mM, less than about 15 mM,less than about 10 mM, less than about 8 mM, less than about 6 mM, lessthan about 5 mM, less than about 4 mM, less than about 3 mM, less thanabout 2 mM, or than about 1 mM. In some embodiments, the concentrationof the quencher in the composition is in the range of about 1 mM to 20mM, about 2 mM to 15 mM, about 3 mM to 10 mM, about 4 mM to 8 mM, orabout 5 mM to 6 mM.

In some embodiments, the composition comprises an additive solution(e.g., a solution described in Table 2, or a solution comprising anycombination of the components described in Table 2). In someembodiments, the composition comprises sodium chloride, adenine,glucose, phosphate, and/or mannitol. In some embodiments, the finalconcentration of chloride ion in the RBC composition (e.g., prior totransfusion) is less than about 500 mM, or about 250 mM, or about 200mM, or about 150 mM, or about 100 mM, about 75 mM, or about 50 mM, orabout 25 mM, or between about 25 and 250 mM, or about 40 and 100 mM, orabout 50 and 75 mM, or about 60 and 70 mM, or about 100 and 200 mM, orabout 125 mM and 175 mM, or about 150 mM.

In some embodiments, the composition is suitable for infusion into anindividual (e.g., a human) after about 2 days, or about 5 days, or about10 days, or about 15 days, or about 20 days, or about 28 days, or about35 days, or about 42 days of storage at 4° C.

In some of these embodiments, the composition comprises a) red bloodcells that are covalently reacted with an electrophilic group of apathogen-inactivating compound (e.g., S-303) on the cell surface and i)have a packed cell volume (PCV) of greater than 60%, and/or ii) have anaverage antibody binding capacity (ABC) of between about 25,000 and70,000 (or about 35,000 and 45,000) and b) a glutathione quencher at aconcentration of less than about 8 mM (or less than 6 mM, or less thanabout 2 mM). In some embodiments, at least 3 log (or at least 1 log) ofa pathogen is inactivated, if present. In some embodiments, thecomposition is suitable for infusion into humans up to 28 or 42 days ofstorage at 4° C.

Kits

In addition to the improved methods of quenching, the present inventionprovides disposable kits for the processing of a red blood cellcomposition, where the processing may be done manually or automatically.In some embodiments, the present invention provides kits comprising thepathogen-inactivating compound, quencher, and/or base used in the eachof the methods described herein. In some embodiments, the kit providesfresh solution (such as buffer for resuspension of the cells) for usefollowing decreasing of the quencher concentration described herein.

In some embodiments, the kit comprises S-303, including any saltsthereof and neutralized glutathione, including any salts thereof. S-303may be in solid form or in solution. Similarly, the neutralizedglutathione may be in solid form or in solution. These solids orsolutions may further comprise acceptable excipients, adjuvants,diluents, or stabilizers. In some embodiments, S-303 is thehydrochloride salt and the neutralized glutathione is neutralized withabout 1 equivalent of sodium hydroxide. In some embodiments, S-303 andneutralized glutathione are in solid form and the kit further comprisesa suitable solution for dissolving the S-303 and a suitable solution fordissolving the neutralized glutathione. In some embodiments, theinvention provides a kit comprising a pathogen-inactivating compound, aquencher and a solution for dissolving the quencher, wherein thesolution neutralizes or partially neutralizes the quencher. The methodsand kits discussed herein encompass any suitable pharmaceuticalformulation of the pathogen-inactivating compound and quencher, whichcan be formulated as a mixture or separately. Pharmaceuticallyacceptable formulations are known to those skilled in the art, andexamples of suitable excipients, adjuvants, diluents or stabilizers canbe found, for example, in (Gennaro, ed., Remington's The Science andPractice of Pharmacy, 20^(th) edition, Lippincott Williams & Wilkins.The invention also includes the resulting compositions of the methodsdescribed above, comprising red blood cells, a pathogen-inactivatingcompound and quencher as described above, wherein the composition is ina suitable pH range to effect improved quenching of thepathogen-inactivating compound.

In another aspect, the invention provides a kit useful, e.g., fortreating red blood cell compositions to inactivate pathogens, comprisinga pathogen-inactivating compound comprising a nucleic acid bindingligand and a functional group which is, or which forms, an electrophilicgroup (including any salt thereof) a quencher comprising a thiol group(including any salt thereof), and about 0.75 to about 1.25 equivalentbase, wherein an equivalent means a molar amount that is equivalent tothe molar amount of quencher in the kit. In some embodiments, the kitcomprises about 1 equivalents of a suitable base.

In still another aspect, the invention provides a kit for treating redblood cell compositions to inactivate pathogens, comprising a nucleicacid binding ligand and a functional group which is, or which forms, anelectrophilic group (e.g., S-303), including any salt thereof, aneutralized quencher comprising a thiol group (e.g., neutralizedglutathione), including any salt thereof, and optionally fresh solution(such as buffer for resuspension of the cells) for use followingdecreasing of the quencher concentration described herein. In someembodiments, the solution is an additive solution, diluent solution,and/or a treatment solution described herein (e.g., SAG-M, AS-5, or anysolution described above or in Table 2, 3, and/or 4).

EXAMPLES, MATERIALS, & METHODS

The invention is further illustrated by the following non-limitingexamples.

Example 1: Organism Preparation

Examples, Materials, & Methods

Bacterial and viral strains used for these studies were clinicalisolates obtained from either the California Department of HealthServices or the American Type Culture Collection.

Bacteria: Frozen working stocks of bacteria were inoculated into a 500mL flask containing a mixture of 50% yeast extract media without addedglucose and 50% fetal bovine serum. The flasks were incubated overnightin a shaking waterbath set at 37° C. Gram positive bacteria were spikedinto the blood product directly from the overnight culture. Theovernight cultures of (Gram negative bacteria were further subculturedby a 1:1000 dilution into fresh culture medium and incubated as aboveuntil they reach log phase as determined by optical density. This logphase growth was spiked into the blood product for PI experiments.

Viruses: Cell free viral stocks were prepared using the appropriate celllines for each virus. These stocks were frozen at −80° C. until theywere thawed and spiked directly into the blood product for PIexperiments.

Example 2: Preparation of RBC Units

Blood was received at Cerus as 450 mL or 500 mL units of whole bloodeither on the day of collection or up to 3 days after collection. Inmost cases the whole blood was leukofiltered before being processed intoRBC units. Occasional units could not be successfully leukofiltered(e.g., blood from donors with sickle trait) and these units were usedwithout leukofiltration for PI studies of organisms that are not knownto survive inside white blood cells.

After leukofiltration, the blood was centrifuged and the plasma wasexpressed. The desired RBC additive solution, such as AS-3 (Nutricel),was then added and the resulting RBC unit was either used immediately orstored at 4° C. until use.

Example 3: Pathogen Inactivation (PI)

The PI process involves inoculation of RBC units with a culture of theorganism to be tested. The typical target input titer of organisms inthe RBC units was approximately 10⁶ cfu or pfu/mL of RBC. In most casesthe organism volume (including any culture medium) was approximately 1%of the RBC unit volume and was not typically greater than 10%. Toevaluate inactivation of lower, more physiologically relevant, levels ofbacteria, inputs from 10 to 10⁵ cfu/unit were used. For low level inputstudies two RBC units were pooled, spiked, then split into a Test unitthat was treated as described herein and a Control unit to which onlyquencher (e.g., GSH) was added (no pathogen inactivator, e.g., S-303)and which was kept under the same temperature conditions as the Testunit.

After addition of the organism into the RBC unit, the unit was mixed bygrasping the ends of the container and moving the ends 10 times in afigure eight, or bicycle pedal, motion.

The contaminated RBCs were then transferred to the mixing container ofthe RBC PI process disposable set. The set consists of a series ofplastic containers and ports connected by plastic tubing. The mixingcontainer was a dual-port 600-mL capacity PL1813 plastic container.Connected to each of the ports was a Y-tubing set with Luer-adaptedpediatric filters attached to one lead. The unused lead on one portconnects to another 600-mL capacity PL1813 plastic container (IncubationContainer). The remaining unused lead was the line used to connect theoriginal RBC unit.

The dosing solutions were prepared and added to the units as follows: A600 mM (Glutathione (GSH) solution with 1 equivalent of NaOH wasprepared by dissolving 2.8 g of GSH in ˜12 mL of 0.9% saline and 0.9 mLof 10 N NaOH. The appropriate volume of GSH solution was drawn into a 20mL capacity syringe. The volume used was typically 10 mL of GSH solutionper 280 mL of RBC, plus 2 mL line loss to generate 20 mM GSH in thedosed RBC unit. The syringe containing GSH was attached to the mixingcontainer using the filtered lead that shares a Y fitting with the leadconnected to the incubation container. The unit was placed on a rockerto facilitate top to bottom mixing during addition of dosing solutions.The GSH was added to the unit while the unit is mixing on the rocker.The unit was then mixed manually using the figure eight mixing methoddescribed above. After addition of GSH the units were allowed to rest atroom temperature for 5 minutes.

After the rest period, a small sample of RBCs were removed and culturedto determine the pre-treatment titer. Standard plate assays were usedfor bacterial samples and cell culture assays were used for viruses.

A 6 mM amustaline hydrochloride (S-303) solution was prepared bydissolving 46 mg S-303 in ˜15 mL of 0.9% saline. The appropriate dose ofS-303 solution was drawn into a 20 ml syringe. The volume used wastypically 10 mL of S-303 solution per 280 mL of RBC, plus 2 mL line lossto generate 0.2 mM S-303 in the dosed RBC unit. The unit was then mixedmanually using the figure eight mixing method as described above. Thetreated RBC unit was incubated at room temperature for a minimum of 3hours after addition of S-303 to ensure completion of pathogeninactivation prior to sampling for post-treatment titer.

At 3 hours post-PI, samples were removed and cultured, as describedabove, to determine post-treatment titer. For studies evaluatinginactivation of low-level bacterial input, both the Test and Controlunits were incubated at RT for ˜20 hours post-treatment and thenincubated at 37° C. overnight. Following 37° C. incubation, samples wereremoved from each treated Test unit and from the identical untreatedControl unit and cultured to obtain a qualitative assessment ofbacterial titer. The untreated Control unit exhibited growth.

Log reduction for each unit was determined by taking the log of theratio of pre-treatment titer to post-treatment titer, where titers wereexpressed as 10×cfu or pfu/mL.

Example 4: RBC In Vitro Function Experiments

Human RBC units were prepared in additive solutions, such as AS-3(Nutricel), according the manufacturer's instructions. RBC units weretreated with various concentrations of GSH to achieve finalconcentrations ranging from 2 mM to 30 mM. In some cases the GSH was pHadjusted with 1 or 2 base equivalents with either sodium bicarbonate orsodium hydroxide prior to treatment. Following treatment with GSH theRBC were treated with S-303, dissolved with 0.9% sodium chloride, toachieve a concentration of 0.2 mM S-303 in the RBC, or mock dosed with0.9% sodium chloride. After treatment, units were incubated for 20 h at20-25° C. Post incubation some units were centrifuged at 6 min, 21° C.,4100×g, supernatant expressed and 100 mL of fresh additive solution wasadded to the RBC. All units were placed at 4° C. for storage. Untreatedcontrols were placed at 4° C. after being prepared in additive solution.

In vitro function was assayed at various time points throughout thecourse of storage. Extracellular pH at 37° C. was determined bymeasuring the pH of RBC from each unit in a Chiron Diagnostics Blood Gasanalyzer. Total ATP was measured using a luciferase based enzymaticassay or a protocol described by Beutler (1984). Cell free supernatantswere prepared to evaluate extracellular potassium, glucose, and lactate.Extracellular Potassium was determined by measuring the K⁺ content ofcell free supernatant using a Chiron Diagnostics Na/K analyzer (model#614) or similar analyzer. Extracellular glucose and lactate wereevaluated on a NexCT analyzer. Red cell indices were collected using theAdvia hematology analyzer (Siemens).

RBCs were washed thrice in 0.9% sodium chloride and incubated a minimumof 1 h at RT prior to analysis for osmotic fragility and densityprofiles. The method used for osmotic fragility is outlined by Beutleret al., 1982, (Blood Journal 59:1141-1147) and was modified for a96-well format (Lew et al., 2003, Blood 101:4189-4194). Densitydistribution curves were obtained according to Danon and Marikovsky,1964 (J Lab Clin Med 6:668-674), using phthalate esters inmicrohematocrit tubes.

Example 5: Quantification of Pathogen-Inactivating Compound Binding toRBC Surface

The level of acridine bound to the RBC surface was detected with asensitive fluorescence-activated immune flow cytometric assay (IFC)using a mouse anti-acridine monoclonal antibody conjugated toallophycocyanin (APC) and a FACS-Caliber flow cytometer (BDBiosciences). Briefly, RBCs were washed thrice in 0.9% saline andresuspended to a 4% hematocrit in flow incubation buffer (HBSS, 1% BSA,0.1% NaN₃, 1 mM EDTA, 3% BSA). Next, mouse monoclonal anti-acridineantibody conjugated to APC was added and incubated for 30 minutes at 4°C.; cells were washed in flow wash buffer (HBSS 1% BSA, 0.1% NaN₃, 1 mMEDTA) resuspended in the same buffer and a total of 30,000 events wereevaluated in the FACS-Caliber at an appropriate gating. Quantificationof numbers of S-303 molecules bound to the cell surface of human RBC(ABC) was performed using Quantum Simply Cellular bead kits (BangLaboratories, Inc; Fishers, Ind.).

Example 6: Glutathione pH Effects on Immediate and Storage-Related RBCHydration and Function

RBC units were treated with S-303 (0.2 mM) and GSH (20 mM) pH-adjustedwith NaOH (varying base equivalents (b.e.)) to potentiate GSH quenching.The pH of tested dosing solutions was 2.9, 4.5, and 8.9 for 0 b.e., 1b.e., and 2 b.e., respectively. Following treatment, RBCs were stored at4° C. and assayed periodically for extracellular pH, glucose, lactate,potassium, total ATP and hemolysis. RBC physical parameters (MCV, MCH,MCHC, HDW etc) were measured by optical flow cytometry, osmoticfragility measurements were performed as per Beutler et al., (1982) withmodification by Lew et al., (2003).

Exposure of RBC to alkaline GSH resulted in decreased Hct with immediateand sustained decreased osmotic fragility and increased RBC density(e.g., see FIG. 1). This immediate dehydration was corrected bydecreasing the base equivalents, resulting in a lower pH of the GSHsolutions (see Table 5). Although immediate dehydration was ameliorated,osmotic fragility of RBCs continued to be changed (Table 6; furtherExamples in FIGS. 2 and 3) by the presence of GSH in a concentrationdependent way. Measurement of MCHC and the distribution of MCH usingoptical flow cytometry (Adiva hematology analyzer, Siemens) correlatedwith changes in osmotic fragility and density. The dehydration was S-303independent as the effect was also demonstrated by pH and GSH in theabsence of S-303.

TABLE 5 Effect of sodium hydroxide base adjusted levels of GSH on RBChydration immediately post-treatment MCF* Hct MCHC Base Level (mOsm) (%)(g/dL) Blood pH Untreated 160 62 31 6.743 Mock dosed Not Done 56 336.759 Dosed (0 b.e.) 173 59 31 6.206 Dosed (1 b.e.) 156 52 36 6.713Dosed (2 b.e.) 142 47 42 7.180 *MCF (Median Corpuscular Fragility) =osmolarity at which 50% of hemolysis occurs

TABLE 6 Effect of GSH base equivalents on RBC hydration and functionduring storage Base MCF* Hct MCHC ATP Glucose Lactate Time** Level(mOsm) (%) (g/dL) Blood pH (μmoles/gHb) (mM) (mM) 20 hr Untreated 158 61± 1 30 ± 1 6.749 ± 0.011 5.35 ± 0.41 11.6 ± 0.1 0.9 ± 0.7 Dosed 156 52 ±1 32 ± 1 6.644 ± 0.034 6.45 ± 0.37 14.5 ± 0.7 0.2 ± 0.0 (1 b.e.) Dosed145 46 ± 1 35 ± 1 7.173 ± 0.020 8.17 ± 0.50 12.8 ± 0.3 0.1 ± 0.1 (2b.e.) 14 Untreated 158 60 ± 1 31 ± 0 6.617 ± 0.020 4.78 ± 0.62  9.8 ±1.3 3.7 ± 0.3 Days Dosed 152 51 ± 1 34 ± 1 6.486 ± 0.019 4.47 ± 0.70 9.4 ± 0.4 2.4 ± 0.6 (1 b.e.) Dosed 146 47 ± 1 36 ± 1 6.651 ± 0.035 5.58± 0.36  6.2 ± 0.9 5.7 ± 0.5 (2 b.e.) 35 Untreated 159 61 ± 3 31 ± 16.477 ± 0.039 3.52 ± 0.37 11.6 ± 0.3 10.6 ± 2.0  Days Dosed 148 52 ± 134 ± 1 6.423 ± 0.033 2.77 ± 0.49 14.5 ± 0.9 20.6 ± 1.6  (1 b.e.) Dosed147 49 ± 1 35 ± 1 6.489 ± 0.030 3.937 ± 0.51 12.8 ± 0.5 10.1 ± 1.0  (2b.e.) *MCF (Median Corpuscular Fragility) = osmolarity at which 50% ofhemolysis occurs **Days post-dosing. Blood dosed at 5 days old.

RBC hydration changes correlated with GSH, pH and concentration, but didnot correlate with S-303 or biochemical assays (ATP, lactate, glucose)routinely used to assess RBC function. Limiting exposure to high pH ofGSH prevented the initial dehydration effect. Limiting continuedexposure of high levels of GSH prevented the storage dehydration effect.These studies show that assessment of the hydration status of storedRBCs should be included as a predictor of RBC quality as substantialchanges in hydration had no effect on conventional criteria but may havecontributed to the moderate change in red cell life span.

Example 7: Improved Quenching Method with Subsequent Decrease inQuencher Results in Decreased RBC Dehydration Following Storage

RBC units were treated with S-303 (0.2 mM) and GSH (20 mM) pH-adjustedwith 1 equivalent NaOH to potentiate GSH quenching. Following treatmentwith GSH the RBC were treated with S-303, dissolved with 0.9% sodiumchloride, to achieve a concentration of 0.2 mM S-303 in the RBC, or mockdosed with 0.9% sodium chloride. After treatment, units were incubatedfor 20 h at 20-25° C. Post incubation some units were centrifuged at 6min, 21° C., 4100×g, supernatant expressed and 100 mL of fresh additivesolution was added to the RBC. All units were placed at 4° C. forstorage. Untreated controls were placed at 4° C. after being prepared inadditive solution. RBC osmotic fragility measurements were performed asper Beutler et al., (1982) with modification per Lew et al., (2003).

Prolonged exposure of RBC to high concentrations of GSH resulted inincreased RBC density and decreased osmotic fragility (e.g., see Table5, FIGS. 3 and 4). This storage-related dehydration was corrected byremoving the GSH prior to RBC storage (e.g., see FIGS. 4 and 5). Thetime and GSH concentration dependent dehydration was S-303 independentas the effect was also demonstrated by GSH in the absence of S-303(e.g., see FIG. 6).

RBC hydration changes correlated with GSH. Limiting exposure to highconcentrations of GSH by exchanging the treatment solution for freshadditive solution prevented the storage induced dehydration effect.

Example 8: Pathogen Inactivation for Improved Quenching Methods

Leukoreduced RBC units with a hematocrit of approximately 60% wereprepared in AS-3 storage medium. RBC units were inoculated withapproximately 6 logs/mL of viable organism, and an aliquot was removedto serve as the untreated, input control. GSH in a solution of 1equivalent NaOH was added to the inoculated units to a finalconcentration of 20 mM and mixed well. S-303 was added to a finalconcentration of 0.2 mM and the units were again mixed well andincubated at 20 to 25° C. for three hours. Following incubation, sampleswere removed and assayed to detect residual viable organisms. Controlsamples were titered immediately after preparation and again after the3-hour incubation period. At least two replicates were performed foreach organism.

With the exception of Pseudomonas, the Gram negative, Gram positive andone example virus were effectively inactivated by treatment with GSHneutralized with 1 equivalent of NaOH compared to neutralization with 2equivalents of NaOH (see Table 7).

Pathogen inactivation of Pseudomonas aeruginosa using a totalinoculation titer of up to 4.4 logs per RBC unit resulted in completeinactivation.

TABLE 7 Pathogen inactivation data for improved quenching conditions vs.previous conditions. Log Reduction Log Reduction GSH (20 mM), 1 b.e. GSH(20 mM), 2 b.e. S. aureus 6.0 ± 0.3 (n = 4) 6.4 S. marcescens 3.7 ± 0.4(n = 3) 4.8 Y. enterocolitica 5.0 ± 0.4 (n = 4) 4.6 E. coli 5.9 ± 0.8 (n= 4) 6.5 P. aeruginosa* 1.2 ± 0.4 (n = 3) 1.8 VSV >5.9 4.2

Example 9: Surface-Bound Acridine Levels for Improved Quenching Methodswith Exchange Step

The method described in Example 5 was used to determine theanti-acridine antibody binding capacity to red blood cells treated with20 mM GSH neutralized with 1 equivalent of NaOH and 0.2 mM S-303. Theantibody binding capacity measured across several RBC preparations wasapproximately 39,000 per red cell (see FIG. 7). This level of bindingcompares to 18,407±1195 ABC when RBC are treated with 20 mM GSHneutralized with 2 equivalents of NaOH and 123,714±5123 ABC when RBC aretreated with 2 mM acidic GSH.

Example 10: Pathogen Inactivation with S-303 Treatment at VariableHematocrit (Hct)

RBC units, from 450 to 500 mL WB collections, were prepared withoutadditive solution (80% spun hematocrit (Hct)) or in additive solution(60% Hct). The RBC were leukofiltered before treatment unless otherwiseindicated. Test units at 40% Hct were diluted with a diluent solution.RBC units were inoculated with either a high level input of ˜10⁶organisms/mL or a low level input from 10 to 10⁵ organisms per unit. Forthe high level input, a control sample of 28 mL was removed prior toS-303 treatment. For the low level bacterial input, Test and Controlunits were prepared by pooling and splitting full RBC units and theControl unit was inoculated with ˜10 organisms per unit. Test units with80% and 60% Hct were treated with 200 μM S-303 and 20 mM GSH,neutralized with one base equivalent of sodium hydroxide (1 b.e.). Testunits with 40% Hct were treated with 130 μM S-303 and 13 mM GSH (1b.e.). The Control samples or units were treated with either 20 mM GSHor 13 mM GSH (1 b.e.) based on Hct. For units with high level input,Control samples were assayed for viable organisms at the time the Testunit was treated. After 3 hours of incubation at RT both the Test unitsand Control samples were assayed for viable organisms, which werequantified by growth on rich agar plates (bacteria) or by plaque assayon Vero cells (VSV). For units with low level input, the Control andTest units were incubated at RT for 20 hours and then at 37° C. for ˜20hours. Samples were then plated to detect bacterial growth. Results areshown Table 8.

TABLE 8 Pathogen inactivation data for samples of varying hematocritvalues. 80% 60% 40% Organism Hematocrit Hematocrit Hematocrit High LevelInput Mean Log₁₀ Reduction^(a) (N = 2) Yersinia enterocolitica 3.4^(b)4.9 6.1 Escherichia coli 3.8^(b) 6.1^(c) 6.6 Serratia marcescens 4.4^(b)4.5 3.1 Staphylococcus aureus ≧5.8^(b) 6.7 >7 Vesticular stomatitis>6.2^(b) >5.9 5.9 virus (VSV) Low Level Input Full Unit Inactivation^(d)(log₁₀) Pseudomonas aerugenosa ≧2.7 ≧2.5 ≧2.5 ^(a)Log reduction iscalculated as Log (Untreated titer/Post-treatment titer), with titerexpressed as 10^(x)/mL ^(b)Pathogen reduction without leukofiltration^(c)n = 3 ^(d)In all cases, control units at the lowest input level werepositive for bacterial growth

Example 11: RBC Hydration Following S-303 Treatment at VaryingHematocrit (Hct)

RBCs were prepared from leukoreduced whole blood at 40% or 60% Hct,measured by spun hematocrit, in additive solution and at 80% Hct aspacked red cells. Units were treated with GSH (sodium salt, BioMedicaFoscama, Italy) and S-303 at a final concentration of 20 mM and 0.2 mMrespectively. All treated units were incubated up to 20 hours at RT. Thetreatment solution was replaced with SAG-M and units were adjusted to60% Hct for storage at 4° C. Control RBC units were prepared in SAG-Mand stored at 4° C. All units were assayed periodically for physicalparameters; MCHC was measured manually, osmotic fragility measurementswere performed by standard methods (Beutler et al., and Lew et al.,2003). Median corpuscular fragility (MCF) was defined as the NaClconcentration at which 50% of RBCs were hemolysed. Change in MCF is anindex of surface to volume ratio (S/V) and hydration of RBC duringstorage. After approximately 6 weeks of storage all treated units hadMCF values comparable to untreated controls, regardless of Hct at timeof treatment. MCHC, another index of RBC hydration, was similar betweenTest and Control units at the end of storage. Results are shown in Table9. Storage of treated RBC for up to 6 weeks did not significantly alterthe RBC hydration and S/V over a wide range of Hct used in routinepractice for preparation of RBC concentrates.

TABLE 9 Hydration data for samples of varying hematocrit values. MCF(mOsm) MCHC (g/dL) (n = 2 ) (n = 2) 20 h post Post 20 h post Post dosingstorage dosing storage 40% HCT 156 152 33 31 80% HCT 158 156 32 30 SAG-M156 158 32 30 Control 60% HCT 151 148 33 33 SAG-M 150 154 32 32 Control

Example 12: In Vitro Quality of Stored RBCs Following S-303 Treatment atVariable Hematocrit (Hct)

RBCs were prepared from leukoreduced whole blood at 40% or 60% Hct,measured by spun hematocrit, in additive solution and at 80% Hct aspacked red cells. Units were treated with GSH (sodium salt) and S-303 ata final concentration of 20 mM and 0.2 mM respectively. All treatedunits were incubated up to 20 hours at RT. The treatment solution wasreplaced with SAC-M and units were adjusted to 60% Hct for storage at 4°C. Control RBC units were prepared in SAG-M and stored at 4° C. In vitrofunction was assayed pre- and post-treatment and at regular intervalsfor up to 6 weeks storage. Parameters assessed for in vitro RBC functionincluded pH, total ATP, hemolysis, and extracellular potassium, glucoseand lactate. After approximately 6 weeks (Day 38 to Day 44) of storage,all Test units had total ATP levels greater than 2 μmol ATP/g Hb and thehemolysis and MCHC were comparable to Control units, regardless oftreatment Hct. Throughout storage the Test unit extracellular glucosewas higher than that of Control units for 40% and 60% Hct, whereas unitswith 80% Hct were more similar to Control. Extracellular lactate waslower in all Test units, regardless of Hct, compared to Control. At theend of storage, extracellular K⁺ was slightly lower in Test units thanControl for 40% and 60% Hct units whereas the 80% Hct units werecomparable to Control. The pH of all Test units was similar to Controlthroughout storage. Hemoglobin yield from the process, regardless oftreatment Hct, met AABB requirements. The activity of all metabolicparameters was similar to Control after S-303 treatment over a widerange of Hct throughout 6 weeks of storage. Results are shown in Table10.

TABLE 10 Metabolic parameters for pathogen inactivation of RBC atvarying hematocrit values. ATP MCHC Glucose (μmol/gHb) % Hemolysis(g/dL) (mmol/L) (n = 2) (n = 2) (n = 2) (n = 2) Test 40% HCT 3.28 (D 38)0.2% (D 44) 31 (D 44) 11.8 (D 44) Test 80% HCT 3.42 (D 38) 0.3% (D 44)30 (D 44) 7.7 (D 44) SAG-M 3.35 (D 38) 0.3% (D 44) 30 (D 44) 6.3 (D 44)Control Test 60% HCT 2.96 (D 42) 0.2% (D 42) 30 (D 42) 9.1 (D 42) SAG-M3.19 (D 42) 0.3% (D 42) 30 (D 42) 5.1 (D 42) Control

Example 13: In Vitro Function and Pathogen Inactivation of Diluted RBCs

SAG-M RBC units were prepared from leukoreduced whole blood units from500 mL collections. For RBC function studies, SAG-M RBC units werepooled by ABO type, and split for matched Test and Control units. Priorto treatment, 150 mL, of diluent solution comprising 28.8 mM mannitol,1.3 mM adenine, 16.2 mM sodium phosphate, 20 mM sodium citrate, pH 7.5was added to Test units. Test units were treated with a GSH sodium saltand S-303 at a final concentration of 20 mM and 0.2 mM respectively.Test units were incubated up to 20 hours at room temperature (RT). AfterRT incubation, units were centrifuged and the supernatant was exchangedwith 100 mL, of SAG-M which was added prior to storage at 4° C. ControlRBC units were prepared in SAG-M and stored at 4° C. All units wereevaluated over approximately 6 weeks of storage at 4° C. by sampling atvarious timepoints. For RBC pathogen inactivation studies, the SAG-M RBCunits were split in half and the RBC units were inoculated withpathogens prior to the addition of the treatment solution and GSH. Afteraddition of the treatment solution and GSH, a control sample (5 mL to 7mL) was removed from the unit to determine input pathogen titer and theremaining unit was treated with S-303. Treated units were sampled forresidual viable pathogen titer after a 3 hour static incubation atambient temperature.

In vitro metabolic and physical indices were evaluated at various timepoints throughout storage with in vitro assays. Extracellular pH at 37°C. was measured in a Siemens Diagnostics Blood Gas analyzer. Total ATPwas measured using a luciferase-based enzymatic assay. Cell-freesupernatants were prepared to evaluate extracellular potassium (K⁺),glucose, and lactate. Extracellular potassium was determined bymeasuring the K⁺ content of cell-free supernatant using a EasyLyte® Na/Kanalyzer. Extracellular glucose and lactate were evaluated on a NexCT™analyzer. Mean corpuscular hemoglobin concentration (MCHC) and spunhematocrit were measured manually. Osmotic fragility measurements wereperformed by standard methods (Beutler et al., and Lew et al., 2003).Median corpuscular fragility (MCF) was defined as the NaCl concentrationat which 50% of RBCs were hemolysed.

For bacterial inactivation studies, RBC were inoculated withapproximately 6.5 log cfu/mL E. coli, S. marescens, S. aureus, Y.enterocolitica, or P. aeruginosa. For viral inactivation studies, RBCwere inoculated with approximately 4.1 log pfu/mL to 6.4 log pfu/mL,depending on the virus. GSH dissolved in saline was added to the unit toa final concentration of 20 mM. Bacterial titers were determined byenumeration of colony-forming units (cfu) on agar plates and viraltiters were determined by enumeration of plaque-forming units (pfu) onappropriate cell lines. Untreated samples were serially-diluted beforeenumeration. Treated samples were not diluted prior to enumeration oftiters.

The results shown in the Tables 11 and 12 below demonstrate acceptableRBC metabolic function and physiological parameters over the course ofthe storage duration and acceptable pathogen inactivation.

TABLE 11 Hydration and metabolic parameters for pathogen inactivation ofdiluted RBC units. Days Post MCF Hct MCHC ATP Glucose Lactate DonationTreatment (mOsm) (%) (g/dL) Blood pH (μmol/gHb) (mM) (mM) 1 Untreated ND54 ± 2 32 ± 0 6.982 ± 0.015 4.50 ± 0.87 34.2 ± 1.3  3.6 ± 0.2 1.8Untreated 153 ± 3 54 ± 3 33 ± 1 6.944 ± 0.015 4.39 ± 0.41 33.1 ± 1.9 4.8 ± 0.1 Treated 152 ± 2 57 ± 1 32 ± 1 6.837 ± 0.016 7.05 ± 0.91 29.4± 1.1  3.9 ± 0.2 7-8 Untreated 154 ± 2 54 ± 3 32 ± 1 6.823 ± 0.021 4.87± 0.45 30.4 ± 1.0 10.3 ± 0.6 Treated 150 ± 2 56 ± 1 32 ± 1 6.701 ± 0.0246.23 ± 0.69 26.3 ± 1.1  7.9 ± 0.5 21-23 Untreated 153 ± 3 55 ± 3 31 ± 16.623 ± 0.022 4.30 ± 0.83 24.5 ± 1.8 18.5 ± 1.4 Treated 148 ± 2 56 ± 132 ± 1 6.526 ± 0.028 4.53 ± 0.99 22.2 ± 0.6 14.1 ± 1.3 35 Untreated 155± 2 54 ± 3 32 ± 1 6.532 ± 0.014 3.31 ± 0.28 21.6 ± 1.3 25.2 ± 0.7Treated 152 ± 2 55 ± 1 33 ± 1 6.429 ± 0.016 3.90 ± 0.49 19.7 ± 0.6 18.9± 1.1 N = 4

TABLE 12 Pathogen inactivation data for samples of diluted RBC units.Average Log Kill Bacteria (n = 4) S. marcescens 4.20 E. coli = 6.69 S.aureus 4.15 Y. enterocolitica = 6.57 P. aeruginosa 3.35

What is claimed is:
 1. A composition comprising: (a) red blood cells,wherein the red blood cells have covalently reacted with anelectrophilic group of a pathogen-inactivating compound; (b) a quenchercomprising a thiol group that is capable of reacting with the pathogeninactivating compound; and (c) a final additive solution; wherein thered blood cells have a Median Corpuscular Fragility value greater than150 mOsm after 35 days at 4° C., wherein the red blood cells have aPacked Cell Volume of greater than 50%, and wherein the composition issuitable for infusion into a subject.
 2. The composition of claim 1,wherein the electrophilic group is selected from the group consisting ofa mustard, a mustard intermediate, and a mustard equivalent.
 3. Thecomposition of claim 1, wherein the electrophilic group comprises anaziridine, an aziridinium ion, a mono- or bis-(haloethyl)amine, a mono-or bis-(mesylethyl)amine, or a mono- or bis-(tosylethyl)amine.
 4. Thecomposition of claim 1, wherein the electrophilic group has covalentlyreacted with the cell surface of the red blood cells.
 5. The compositionof claim 1, wherein the pathogen-inactivating compound is β-alanine,N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester.
 6. Thecomposition of claim 1, wherein the quencher comprises cysteine or aderivative of cysteine.
 7. The composition of claim 6, wherein thequencher is glutathione or a pharmaceutically acceptable salt thereof.8. The composition of claim 1, wherein the quencher is at aconcentration of less than about 10 mM.
 9. The composition of claim 1,wherein the quencher is at a concentration of less than about 8 mM. 10.The composition of claim 1, wherein the quencher is at a concentrationof less than about 6 mM.
 11. The composition of claim 1, wherein the redblood cells have a Packed Cell Volume of greater than 55%.
 12. Thecomposition of claim 11, wherein the red blood cells have a Packed CellVolume of greater than 60%.
 13. The composition of claim 1, wherein thered blood cells have an average antibody binding capacity (ABC) of lessthan about 50,000.
 14. The composition of claim 13, wherein the redblood cells have an average antibody binding capacity (ABC) of less thanabout 40,000.
 15. The composition of claim 1, wherein the red bloodcells have an average antibody binding capacity (ABC) of between about25,000 and 70,000.
 16. The composition of claim 15, wherein the redblood cells have an average antibody binding capacity (ABC) of betweenabout 35,000 and 45,000.
 17. The composition of claim 1, wherein thefinal additive solution comprises one or more of sodium chloride,adenine, glucose, and mannitol.
 18. The composition of claim 17, whereinthe final additive solution further comprises phosphate.
 19. Thecomposition of claim 17, wherein the final additive solution furthercomprises citrate.
 20. The composition of claim 1, further comprisingone or more degradation products of the pathogen-inactivating compound.21. The composition of claim 20, wherein the one or more degradationproducts of the pathogen-inactivating compound comprise one or morehydrolysis products of the pathogen-inactivating compound.
 22. Thecomposition of claim 20, wherein the one or more degradation products ofthe pathogen-inactivating compound comprise an acridine.
 23. Thecomposition of claim 1, wherein the final additive solution is anadditive solution selected from the group consisting of AS-1, AS-3,SAG-M, Erythrosol, AS-5, PAGGS-M, and MAP.
 24. The composition of claim1, wherein the red blood cells have a Median Corpuscular Fragility valuegreater than 150 mOsm after 42 days at 4° C.
 25. The composition ofclaim 7, wherein the quencher is glutathione monosodium salt.
 26. Thecomposition of claim 1, wherein the pathogen-inactivating compound isβ-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester, andwherein the quencher is glutathione monosodium salt.
 27. The compositionof claim 26, further comprising an acridine degradation product of thepathogen-inactivating compound.